[Objective] hspB, the gene of the key monooxygenase in nicotine degradation from Pseudomonas putida S16, was cloned and expressed in Escherichia coli; protein HspB was purified, and the crystal condition of HspB was studied. [Methods] The gene hspB was amplified from the genomic DNA of Pseudomonas putida S16. Then recombination plasmid pET28a-hspB was expressed in E. coli BL21. Ni2+-NTA His·Bind and gel filtration were used to purify HspB. The preliminary crystal of HspB was screened and optimized by using hanging drop diffusion method. [Results] pET28a-hspB plasmid was successfully constructed and the protein HspB was purified to crystalline purity. Crystal condition of HspB was obtained and the culture condition is 0.2 mol/L NaCl, 0.1 mol/L HEPES pH 7.5, 1.1 mol/L (NH4)2SO4, 4 °C, Seeding. [Conclusion] The construction of the HspB purification system and the study of preliminary crystal of HspB lay a foundation for the research of structure-function relationship and improvement of HspB catalytic efficiency by directed evolution of gene manipulation.