Abstract: In this study, lotus seed starch was used as the raw material for preparing RS3 resistant starch (EHP-LRS3) through thermoacidophilic type Ⅲ pullulan hydrolase synergistic high-pressure heat treatments. The parameters for this preparation process were optimized using a single-factor experimental design and response surface methodology. The morphological and crystal structures of EHP-LRS3 were observed and analyzed using scanning electron microscopy (SEM) and X-ray diffraction (XRD), respectively, and its pro-proliferative effect on Bifidobacterium longum was investigated. According to the results, the optimal conditions for preparing EHP-LRS3 with TK-PUL enzymatic hydrolysis temperature of 80 ℃ were as follows: Treatment of 35.32% lotus seed starch slurry (pH5.00) with 25.00 U/g (lotus seed starch) TK-PUL at 80 ℃ for 12.70 h, followed by high-pressure heat treatment of the mixture at 121 ℃ for 10 min and retrogradation at 4 ℃ for 24 h. An EHP-LRS3 yield of 58.46% was achieved under these optimal conditions. The SEM analysis revealed that the prepared EHP-LRS3 possessed an irregular ravine-like structure. The XRD analysis showed that the resistant starch had a B-type crystal structure, which differed from the A-type crystal structure of lotus seed starch. The pro-proliferative effect of EHP-LRS3 on B. longum was superior to that of RS3 resistant starch prepared using high-pressure heating alone (HP-LRS3). In summary, a high EHP-LRS3 yield was obtained using both TK-PUL and high-pressure heat treatments. The superior pro-proliferative effect of EHP-LRS3 on B. longum was verified. The results provide a theoretical basis for the high-value utilization of lotus seed starch.