Abstract: Pullulanase is one of the key enzymes for the efficient utilization of starch. Therefore, it is of great significance for the industrial application of pullulanase to select high-yield pullulanase strains. In this study, a strain L5 producing pullulanase has been screened from the soil, and the pullulanase activity reached 10.83 U/mL. The strain L5 was identified as the genus of Bacillus by the methods of 16S rDNA sequencing and phylogenetic tree construction. Four high-yield mutants G1 (20.33 U/mL), G2 (19.68 U/mL), G3 (19.31 U/mL) and G4 (22.01 U/mL) were screened from L5 strain by using ARTP technology, and their enzyme activities were increased by 87.7%, 81.7%, 78.3%, and 103.2%,respectively. Then the fermentation medium was optimized by orthogonal experimental design. The optimum fermentation medium composition was as follows: soluble starch 1.5%, dextrin 1.0%, peptone 0.5%, soybean powder 1.5%, Ca²⁺ 0.05%, Fe²⁺ 0.10%, and Zn²⁺ 0.15%. Under the condition of optimum fermentation medium, the pullulanase activity of G4 reached 27.22 U/mL, which was 23.7% higher than control. The enzymatic properties were also studied. The optimum reaction temperature and pH value of pullulanase were 50 ℃ and 6.5 respectively. The activity of pullulanase could be maintained over 80% at 30-60 ℃ for 2 hours and over 60% with the pH value of 5-9 for 4 hours. Hence, the enzyme had good thermal stability.