Abstract: A mutant strain GF-21with high yield and stable inheritance was obtained by double round mutagenesis of ultraviolet-LiCl and microwave-LiCl using Serratia marcescens G3-1 as original strain. The fermentation conditions of GF-21 were investigated using single factor and orthogonal experiment. The result showed that the optimal fermentation medium contained 6 g/L lactose, 10 g/L urea, 1.0 mmol/L KCl and 5 g/L NaCl, 10 g/L colloidal chitin. Under the optimal fermentation medium condition, the chitinase activity was 4.73 U/mL which was 1.09-fold higher than that before optimization and was 4.7-fold higher than original strain. The optimal fermentation conditions were as follows:the initial pH9.0, the temperature 30℃, the shaking speed 220 r/min, the inoculation age 5 h, the inoculum size 10%. The result provided a theoretical foundation for chitinase applications.