More than 95 percent of the working-parent households in the District of Columbia whose family incomes are supplemented by welfare payments will lose those benefits over the next six months because of new eligibility guidelines imposed by the Reagan administration, city officials said yesterday.

James A. Buford, the city's director of human services, said the 3,500 families -- who account for about one of every 12 families in the city on welfare -- will be bumped from the rolls because the guidelines place new limits on the amount of earned income deductions that working welfare parents can claim for child care, clothing, transportation and other work-related expenses.

At the same time that the city will be eliminating those persons because it cannot afford the $3 million to offset the loss of federal reimbursements, it will spend $1.4 million to maintain benefits at present levels for 19- and 20-year-olds in school, mothers in their first six months of pregnancy, and persons on strike, Buford said.

New U.S. guidelines prohibit payments for persons in those categories, Buford said, but Mayor Marion Barry decided this week that it was essential for the city to pick up the cost of these programs.

There are 2,123 persons covered under the 19-20-year-old program receiving an average of $90 a month, and 185 expectant mothers in the early terms of pregnancy, city officials said.

The average monthly stipend that would be lost by the working households is $143. All of the payments are made under the Aid to Families with Dependent Children program, which affects 29,000 city families.

City officials and welfare advocacy groups predicted yesterday that the loss of benefits could have a severe effect on the working welfare families.

"It's a disaster," said City Council member Polly Shackleton (D-Ward 3), chairman of the council's committee on human services.

Brenda Gibbs-Moore, a lawyer with Neighborhood Legal Services, said, "It's a real blow to these people. The earned income guidelines have always been one way for people to work their way off welfare, to receive support for a few months while they get back on their feet."

"Now the government is saying, 'We're going to penalize you for working,' " Gibbs-Moore added. "It's definitely counterproductive because a lot of these people are going to end up back on welfare anyway."

City officials said yesterday that the typical working parent family that would be affected by the new guidelines would consist of a mother with one child in school and another in day care, who works part-time at a minimum wage salary -- receptionist, hospital orderly, nurse's aide or clerk typist, for example.

The mother can now earn up to $9,000 a year and still be eligible for the average monthly supplement of $143. The new guidelines alter a complicated series of earned income deductions that partially determine eligibility.

For instance, under the new provisions there will be a flat $75-per-month cap on the cost of working (clothing, transportation, etc.) plus a $160-a-month ceiling on child care expenditures. There were no ceilings before.

Previous guidelines also permitted unlimited use of an automatic monthly deduction of $30 plus one-third of the total deductions. The new guidelines limit use of the automatic deduction to four months.

Gibbs-Moore said that some of the working parent families removed from the rolls may only be receiving monthly welfare checks of $10 or $20. But the impact still will be severe, she said, because, when they are dropped from welfare, they also will be cut off from Medicaid, which provides them with subsidized health care.

Human services officials said the $3 million the city hopes to save by removing the families from the rolls may never be realized because many may choose to stop working rather than allow their benefits to cease.

"There's absolutely no certainty that there's going to be any savings at all," said Lee Partridge, special assistant to Buford.

District of Columbia officials did not calculate any cost of maintaining welfare for persons on strike because there is no way, they said, of knowing what strikes might take place. "But if we have a strike, we could have problems," Buford said.

Officials also said yesterday that there could be a rash of confusion among the city's welfare population when new federal monthly reporting guidelines take effect starting in November.

The guidelines, based on policies that have been in effect for years in Los Angeles and other cities around the country, will require each family to fill out a monthly form answering a series of questions about any changes in their income or employment status.

"The experience in other states is that there is initial confusion," Partridge said.

In Maryland and Virginia the exact impact of the Reagan requirements is still uncertain.

Maryland expects about 35,000 of its 220,000 recipients to lose welfare and food stamp benefits, according to Dick Lacombe, executive director of the income maintenance division at the state Department of Human Resources.

In Virginia, many of Reagan's changes already are in effect. Women in the first six months of pregnancy and 18-20-year-olds are not currently eligible under Virginia law. The Reagan earned income test is more stringent than Virginia's and will result in some expuslion from the rolls, according to state officials.