Abstract: The aim of this study was to isolate and identify a strain with high nicotine degradation activity in the situation where there are few kinds of nicotine-degrading microorganisms available, and to explore the conditions and characteristics of nicotine degradation, which will provide a new microbiological resource for the biodegradation of nicotine. A strain with high activity of nicotine degradation was obtained by isolation and purification from tobacco field soil. The strain was identified by morphological, physio-biochemical and 16S rRNA gene analysis, and the characteristics of nicotine degradation under different conditions were investigated with dynamic process by HPLC analysis. The optimum condition of nicotine degradation by Stenotrophomonas sp. ZUC-3 was 30 ℃ with 10% inoculum at the initial pH 7.0 and 180 r/min, in which the rate of nicotine degradation by Stenotrophomonas sp. ZUC-3 reached 91.53%. HPLC analysis illustrated the dynamic process and high efficiency of nicotine degradation. The study provides a new microbiological resource for biodegradation of nicotine and has theoretical and practical significance in tobacco industry.