Abstract: In order to acquire fusant with high nicotine-degrading ability，Bacillus amyloliquefaciens T11 and Pseudomonas marginalis were used for parent strains. After using lysozyme to break the cell walls of parent strains， the Bacillus amyloliquefaciens T11 protoplast and heat-inactivated Pseudomonas marginalis protoplast were induced by 40% PEG-6000 for protoplast fusion experiment，then the nicotine-degrading ability of fusant was measured. Through preliminary screening，176 fusion strains were obtained. After rescreening by nicotine selective culture medium，5 fusion strains with higher nicotine-degrading ability were acquired. Genetic stability analysis of the 5 fusion strains and comparision with parent strains’ nicotine-degrading rate were further measured. A better fusant A2 with higher nicotinededrading ability than parent strains was obtained finally，93% nicotine in culture medium was degraded in five days.