• A hyperthermophilic amylopullulanase is recombinantly expressed in E. coli. • The enzyme is able to efficiently improve resistant starch yield from rice starches. • Pregelatinization of starches and enzymatic hydrolysis were combined into one step. A glycoside hydrolase family 57 protein, designated APU_Aquae, from hyperthermophilic bacterium Aquifex aeolicus was expressed in Escherichia coli . APU_Aquae was biochemically defined as a novel bifunctional amylopullulanase, which exhibited efficient activity towards rice starches. It is extremely thermostable with half-life of 7 h at 100 °C. Therefore, APU_Aquae was used to simplify the process of enzymatic debranching treatment in resistant starch type III (RS3) preparation from rice starches. Starch pregelatinization and enzymatic hydrolysis were combined into one step. It took only 7 h, which was much shorter than that of other enzymatic treatments. RS3 content of treated rice starches was 40.5 ± 0.7%. APU_Aquae treatment resulted in more medium branch chains B1 with degree of polymerization (DP) values 13–24, which might be one main reason to promote the RS3 formation. This study provides the new design for the industrial RS3 production.