A one-pot two-enzyme reaction system was developed to produce high-fructose syrup (HFS) containing healthy rare sugar d-allulose from Jerusalem artichoke (JA). Inulin in JA was converted through the cascade reaction of a novel exo-inulinase from Bacillus velezensis (BvInu) and D-allulose 3-epimerase from Ruminococcus sp. (RDAE). BvInu and RDAE were expressed in Bacillus subtilis and successfully secreted into the supernatant, which decrease the production cost and avoid enzyme purification. The optimal temperature and stability of extracellular BvInu significantly increased, thereby promoting the catalytic activity of the two enzymes for the cascade reaction in one pot. Inulin in JA powder was almost completely converted into monosaccharides in the treatment with the optimal ratio of BvInu : RDAE (80 : 40 U/g inulin) at 50 degrees C for 2 h. The ratio of D-glucose, D-fructose, and D-allulose in the product was approximately 1:3:1, and the yield on JA powder was 67%. The system exhibits potential high-valued application of non-grain crops on producing HFS with D-allulose.