The DSCC ad, launched a day after actress Ashley Judd decided against challenging McConnell, charges that he sides with "Washington special interests" against Kentuckians.
The 60-second radio ad is themed after the college basketball championship, and will run statewide from March 28 to April 3.
McConnell's radio ad features clips of his speech at the Conservative Political Action Conference earlier this month, in which he defended conservatism and issued a rallying cry to the gathered Republicans to stand up to President Obama.
"If you believe in your heart that the direction Barack Obama wants to take this country is wrong, it's time to stand up together and punch back," he says in the speech, first reported by local news site Channel 2.
The ad will run statewide on a mid-five-figure buy. according to Channel 2.
Democrats see McConnell as being one of the most vulnerable sitting Senate Republicans up for reelection next year, but they have had trouble finding an opponent to run against him.
Washington Democrats liked Judd, who would have entered the race against McConnell with deep pockets and a high profile. Kentucky Democrats, however, were wary of the Hollywood actress, given her liberal politics and past controversial statements.
Judd's decision not to run shifts attention to Kentucky Secretary of State Alison Lundergan Grimes, who is exploring the possibility but has not yet made any decisions.
Republicans note that Judd is the 10th prominent Democrat in Kentucky to decline a run against McConnell, though she's one of just a handful of serious recruits that have taken themselves out of the race thus far.
Democrats face a tough landscape overall in 2014, as they are defending 20 Senate seats. A number of the seats are in states won by 2012 GOP presidential candidate Mitt Romney, and five of them will be for seats vacated by sitting Democrats.
Making McConnell a viable target would broaden the playing field for Democrats, and DSCC Executive Director Guy Cecil on Thursday previewed the party's likely line of attack.
"Mitch McConnell is the walking, talking embodiment of everything that’s wrong with Washington D.C.," he said in a statement. "He has helped send Kentucky jobs overseas and blocked job training for Kentucky workers. No wonder Mitch McConnell is the nation’s least popular Senator. Kentuckians deserve a leader who fights for them, not the Washington special interests."
--This piece was updated at 12:14 p.m. to reflect McConnell's new ad