Jacquelin, Kieffer and Felipe Aguilar of Colombia all finished on five under par after a testing final day of the event at the Parador de El Saler in Valencia as overnight leader
Marc Warren of Britain slumped to a four-over-par 76 for a share of fourth place on four under.
Jacquelin and Kieffer birdied the third play-off hole to knock Aguilar out of contention and the pair needed a further six attempts before Jacquelin sunk another birdie to add to Tour wins at the Madrid Open in 2005, the 2007 Asian Open and the Sicilian Open two years ago.
Lasting more than two hours, it was the joint-longest play-off on the Tour since the series was created in 1972, matching the 1989 Dutch Open.
"That is very tiring but I am really, really happy," Jacquelin said on the Tour website (www.europeantour.com).
"I could not make any putts (in the playoff) but the last one dropped, which means a lot for me and my family," added the Lyon-born 38-year-old.
"There's another baby coming at the end of November. Every time I get a baby there's a win so I'll have a football team after a few years," he joked.
Kieffer, 22, turned professional in 2010 and has one Challenge Tour title to his name, last year's Gujarat Kensville Challenge in India.