2,3-Butanediol is one of the promising bulk chemicals with wide applications. Its fermentative production has attracted great interest due to the high end concentration. However, large-scale production of 2,3-butanediol requires low-cost substrate and efficient fermentation process. In the present study, 2,3-butanediol production by Klebsiella pneumoniae from Jerusalem artichoke tubers was successfully performed, and various technologies, including separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), were investigated. The concentration of target products reached 81.59 and 91.63 g/l, respectively after 40 h in batch and fed-batch SSF processes. Comparing with fed-batch SHF, the fed-batch SSF provided 30.3% higher concentration and 83.2% higher productivity of target products. The results showed that Jerusalem artichoke tuber is a favorable substrate for 2,3-butanediol production, and the application of fed-batch SSF for its conversion can result in a more cost-effective process.