Harris said she would instead run for re-election to the U.S. House seat she has held for two years.
House Speaker J. Dennis Hastert praised her decision, calling Harris "an effective leader who always puts people before politics."
"I am glad that she plans on continuing her service in the House," he said.
Rumors of a possible Harris Senate run set off shockwaves within the national GOP, who feared her candidacy could distract from the president's re-election efforts in Florida and would unite and energize the Democrat's national fund-raising base.
Several recent polls suggest Harris likely would have won a plurality of the GOP vote in a crowded primary that also includes former Bush Housing Secretary Mel Martinez, former Rep. Bill McCollum, who lost a 2000 open seat bid, state Sen. Daniel Webster and outgoing state House Speaker Johnnie Byrd.
Democrats currently seeking the nomination include former state Education Commissioner Betty Castor, Miami-Dade County Mayor Alex Penelas and Rep. Peter Deutsch.