The 108th meeting between the two sides in all competitions will be the first time that the teams have met in a UEFA competition, adding extra spice to this season's last 16.
Betis have belied their position at the foot of La Liga with their European exploits this term, losing just once in 10 games in the competition in 2013-14.
A 2-0 away win against Rubin Kazan saw Betis progress 3-1 on aggregate into the last 16, while Sevilla, who are unbeaten in their Europa League campaign, overcame Maribor 4-3.
The Estadio Ramon Sanchez Pizjuan will host the first leg and hosts Sevilla have romped to 4-0 and 5-1 wins in their last two home outings against Betis.
November's 4-0 home victory saw jubilant scenes from the home fans, but Sevilla president Jose Castro has called for caution heading into Thursday's match.
"Derbies are different," he said. "If the (league) table is true we are better than Real Betis, but often the lower placed side wins the match. The stakes are high.
"I do not think this tie will be easy or have as many goals as in recent games."
After a run of only one league win since the end September heading into last weekend, Betis looked in danger of surrendering their top-flight status without a fight, but a 2-0 victory over Getafe on Saturday has restored confidence, according to the club's president Miguel Guillen.
The Europa League has been a welcome distraction and Guillen hopes Betis can continue the little bit of momentum that they have created in the competition.
"There have been a series of games that have helped restore confidence," he said on Tuesday. "We have an obligation to try to win every game that comes our way, even more so in the case of a derby.
"It will be a competitive game, but the fact that one team is (performing) better than another in the league is not going to determine anything."
