Abstract: In this paper, the rhizosphere soil of Tongshan tobacco（Zhongyan 100） production field was used as material, three strains of starch debranching enzyme producing strains were obtained by solid identification medium with amylopectin as carbon source, which were numbered XUEX1, XUEX2 and XUEX3 in turn. The enzyme producing activities of the three strains were measured by DNS method. After 32 hours of continuous fermentation, the peak enzyme activity of XUEX3 reached 16.3 U/mL, XUEX1 and XUEX2 did not exceed 9 U/m L.The XUEX3 strain with the highest enzymatic activity was selected as the starting strain for breeding by ultraviolet mutagenesis. The results showed that the three positive mutants, XUEX3-1, XUEX3-2 and XUEX3-3, which had the highest of enzyme production, had significantly increased the activity of isoamylase compared with the original strain, and the HC value of XUEX3-3 was 3.2 times of that of the original strain. In the continuous fermentation of 32 h, the peak value of enzyme production activity of XUEX3-1, XUEX3-2 and XUEX3-3 were 39.7 U/m L,43.1 U/m L and 35.3 U/mL, respectively. Compared with the control, the peak value of enzyme production of XUEX3-2 was 4 hours earlier, and that of XUEX3-1 and XUEX3-3 was 4 hours later. However, after 5 generations of continuous culture, the genetic stability of positive mutants XUEX3-1, XUEX 3-2 and XU-EX3-3 was stable.The results enriched the genetic resource pool of starch debranching strains and provided more options for later application and development.