Abstract: In this study, the effects of the initial pH value, the levels of inoculum and culture temperature on nicotine degradation were investigated in flasks using a novel nicotine-degrading bacterium, Ochrobactrum intermedium DN2. A Box-Behnken experimental design was applied in the design of experiments and in the analysis of the experimental data. When α=0.05, three linear coefficients and all quadratic coefficients were significant. Among the interaction effects, the interaction coefficients of the initial pH value vs. levels of inoculum, levels of inoculum vs. culture temperature were significant, while the interaction coefficient of the initial pH value vs. culture temperature was not significant. The experimental data also allowed the development of an empirical model describing the interrelationship between independent and dependent variables. By solving the regression equation, the optimal values of the variables were determined as follows: the initial pH value of 7.27, level of inoculum of 14.93 g/L, temperature 31.85℃, with the corresponding Y=66.13%, while actual value was 64.82%, indicating that the model is satisfactory and practicable. Under these conditions, process of nicotine degradation in tobacco waste extract was also carried out. The results showed that 83.83% of nicotine was degraded in 36 h, the degradation process of nicotine followed zero-order reaction of Monod model. These results indicate that strain DN2 is of application value in nicotine pollution treatment.