U.S. District Judge Barbara Crabb denied the request from Wisconsin Attorney General J.B. Van Hollen, who has appealed her decision and asked that weddings be halted to reduce confusion.
Hundreds of gay couples have married in Wisconsin's two most populous counties, Milwaukee and Dane, home to the state capital, Madison, since Crabb's ruling, while clerks in many other counties have not issued licenses.
Van Hollen had sought the stay from Crabb on Friday and on Monday asked the U.S. Court of Appeals for the Seventh Circuit to halt gay marriages in Wisconsin until appeals are concluded.
Until then, Van Hollen said in a statement, Wisconsin's ban on gay marriage remains in effect and state and local officials are under a continuing duty to follow the marriage law unless and until the court enjoins that law.
Crabb's rulings have not addressed directly whether county clerks may issue marriage licenses to same-sex couples in the interim.
"I understand defendants' concern that some county clerks have been issuing marriage licenses to same-sex couples since I issued the June 6 decision, but that is not a result of an injunction by this court," Crabb wrote.
More than 280 marriage licenses were issued to gay couples on Friday and Saturday in Milwaukee and Dane counties after Crabb ruled the ban unconstitutional. A steady stream of couples sought licenses Monday in counties around the state.
"His (Van Hollen's) appeal of this case is a colossal waste of taxpayer dollars and I urge him to reconsider," Dane County Executive Joe Parisi said in a statement.
Dane County had issued at least 20 marriage licenses to gay couples on Monday by mid afternoon and planned to continue, Dane County Clerk Scott McDonell said after Crabb's latest ruling.
The office of Milwaukee County Clerk Joe Czarnezki said it had issued more than 35 marriage licenses to gay couples Monday.
Crabb's ruling was the latest in a string of decisions by federal judges who have struck down gay marriage bans in a number of states.
Challenges to state bans gathered momentum last June when the U.S. Supreme Court struck down parts of the federal Defense of Marriage Act, ruling that legally married same-sex couples were eligible for federal benefits.
Not including Wisconsin, same-sex marriage is now legal in 19 states plus the District of Columbia. That number would jump sharply if federal court rulings striking down bans in several states are upheld on appeal.