Abstract: Jerusalem artichoke tuber (JAT) is rich in inulin and it is considered as one of the most important feedstocks for fructooligosaccharides (FOS) production. Direct enzymatic production of functional syrup from fresh JAT can benefit comprehensive processing of Jerusalem artichoke. In this study, the effects of various enzymes (endogenous hydrolases, endoinulinase, endoglucanase, xylanase, polygalacturonase and tannase) on fructooligosaccharide production from JAT were systematically studied and enzymatic process were further optimized. The optimal process was: first loading tannase (0.08 U/g) at 50 °C and pH 5 for 4 h, and then loading endoglucanase (0.1 U/g), xylanase (0.08 U/g), polygalacturonase (0.07 U/g) and endoinulinase (12 U/g) for 8 h. The FOS syrup was obtained by concentrated reaction solution for tow folds. The FOS and total polyphenols in FOS syrup reached 53.72 g/L and 3.11 g/L. The DPPH free radical scavenging rate, hydroxyl radical scavenging rate and total antioxidant capacity of FOS syrup were 82.23%, 30.47% and 2.78 μmol/mL, respectively. Replacing glucose in MRS medium with prepared fructooligosaccharide syrup as sole carbon source, the growth rates of Lactobacillus plantarum, Streptococcus thermophilus and Lactobacillus paracasei increased by 33.33%, 60.47% and 148.15%, respectively, compared with the unenzymatically hydrolyzed Jerusalem artichoke puree as the sole carbon source of MRS medium. The main results of this study provide a theoretical basis for the enzymatic production of prebiotic food from Jerusalem artichoke.