Abstract: 【Objective】 This study aimed to screen efficient nicotine-degrading strains and optimize their fermentation conditions， so as to provide technological supports for harmless treatment of tobacco waste.【Method】 Nicotine-degrading strains were isolated from tobacco-planting soil in Xuchang City， Henan Province using nicotine media and identified by morphological and molecular biological methods. The single factor test combined with orthogonal test design were used to obtain optimal fermentation conditions for nicotine degradation. After inoculating tobacco waste extracts with the strains， the optimal nicotine degradation conditions for tobacco waste were determined using response surface test.【Result】 A nicotine-degrading strain YT5 was isolated from tobacco-planting soil. The strain was identified as Pseudomonas putida by colony morphological observation and phylogenetic tree analysis based on 16S rDNA sequences. The single factor test showed that the YT5 growth and nicotine degradation rate were optimal at fermentation temperature of 30 °C， inoculum amount of bacterial suspension of 4%， initial pH of 6.5-7.0， nicotine concentration lower than 4 g/L， and culture time of 24 h. After optimized by orthogonal experimental design， the optimal fermentation conditions for nicotine degradation of the strain YT5 were temperature of 30 ℃， initial pH of 7.0， inoculum amount of bacterial suspension of 6%， culture time of 24 h， and nicotine degradation rate of strain YT5 could reach 95.79%. Furthermore， after degradable tobacco waste extracts were inoculated with strain YT5， and the optimal fermentation process conditions were determined to be culture time of 29.3 ℃， initial pH of 7.1， inoculum amount of bacterial suspension of 5.1%， under which the nicotine degradation rate of tobacco waste extracts reached 55.54%； effects of culture temperature and initial pH on nicotine degradation rate were extremely significant （P<0.01）， and the interaction between initial pH and inoculum amount of bacterial suspension was significant （P<0.05）.【Conclusion】 A nicotine-degrading strain YT5 is obtained， and the strain is able to degrade nicotine in tobacco waste extracts， which can be used for biodegradation of nicotine in industrial tobacco waste.