With most of the vote counted in the southern US state, the ex-Pennsylvania senator secured 49%, well ahead of the front-runner Mitt Romney, who had 27%.
Mr. Santorum is seeking to close the gap on the ex-Massachusetts governor to become the party’s nominee to challenge Barack Obama’s in November’s election.
Mr. Romney has now won 21 out of 34 contests to Mr. Santorum’s 11.
Former House Speaker Newt Gingrich was trailing in Lousiana’s poll in third place, ahead of Texan Ron Paul – who has yet to win a poll.
“We’re still here. We’re still fighting. We still believe, as this race really shows,” Mr. Santorum said in Wisconsin, where he watched the election results.
He enjoys strong support among conservatives in the south, and he has also picked up backing from Republicans who have peeled away from Mr. Gingrich as he lost momentum.
However, Mr. Romney is widely tipped to regain the advantage when the race moves on in April to Wisconsin, Maryland and Washington DC.
He also received a boost earlier this week when he secured the endorsement of former Florida Governor Jeb Bush.
Mr Bush called on Republicans to “unite” behind the Romney campaign.
Correspondents say the backing of the respected Republican suggests the party establishment could be coalescing around Mr. Romney.
In another boost, Freedom Works, an organization that supports the Tea Party movement, dropped its opposition to Mr. Romney’s candidacy.
Mr. Romney has so far won 563 delegates and Mr. Santorum’s 263, according to an estimate by the Associated Press news agency.
Louisiana’s primary allocated 20 of the state’s 46 total delegates; the rest will be decided at the state party convention in June.
A candidate needs to accumulate 1,144 delegates to the August convention in order to secure the Republican nomination.