Abstract: The conditions for the efficient secretory expression of thermoacidophilic type Ⅲ pullulan hydrolase Tk-PUL in Bacillus subtilis were explored,and the enzymatic properties of recombinant Tk-PUL were preliminarily studied.To identify the signal peptide that guide Tk-PUL efficiently secretory expressed in Bacillus subtilis,a screening library of signal peptides for Tk-PUL was constructed and high-throughput screening method was used. Results showed that,under the guidance of signal peptide AmyE,recombinant Tk-PUL was efficiently secretory expressed in Bacillus subtilis.Tk-PUL was a bifunctional enzyme with a single active site,Tk-PUL possessed both α-amylase and pullulanase activities. The highest α-amylase activity was observed at pH4.5 and 100℃,the corresponding specific activity was 54.08 U/mg,and its half-life at 100℃ was 2 h. The highest pullulanase activity was observed at pH4.5 and 100℃,the corresponding specific activity of 110.39 U/mg,and its half-life at 100℃ was 2 h. This study establishes the foundation for the application of Tk-PUL in starch processing industry.