Abstract: This study aimed to use E. Coli BL21( DE3) to overexpress recombinant chitinase and study the concentration of inducer,time and temperature of expression to optimize the expression. The recombinant chitinase was not only in the soluble protein but also in the inclusion body. The refolding methods by dialysis and Ni-NTA column were used to renature the chitinase and were compared by the content of protein and specific enzyme activity. The effects of the loading protein amount,the flow rate of refolding buffer and the temperature on the refolded chitinase were investigated. The results show that chitinase could be refolded by dialysis and Ni- NTA column. The enzyme activity of chitinase by the affinity chromatography which was1347.7 U/mg was higher than by the dialysis method,but its yield by the affinity chromatography was lower than by the dialysis method. The enzyme activity of refloding increased when the chitinase sample with lower concentration was loaded to the column under lower temperture at the optimal elution rate of 0.4 m L·min-1. The chitinase from the affinity chromatography refolding possessed higher activities to degrade fluorescent substrate.The optimal temperature was 37 ℃ and the optimal p H value was 3.8.