Abstract: Cowpea has a red nodule containing large amounts of bean hemoglobin. The protein is a good natural pigment and can be used as color agent in artificial meat. In this study, a 456 bp sequence of cowpea leghemoglobin Ⅱ (Lb II) was obtained from the NCBI database. Using the pET-15b as the expression vector and E. coli BL21-CodonPlus (DE3)-R-IL as the recombinant expression host, the cowpea Lb Ⅱ was successfully expressed, then preliminarily purified this protein by using the nickel column chromatography and added ascorbic acid as the antioxidant simultaneously. IPTG concentration, temperature and time were used as the independent variables and Lb Ⅱ expression as the dependent variable to univariate experiments. The results showed that the highest yield of recombinant Lb Ⅱ protein was obtained at a final IPTG concentration of 1.0 mmol/L and 25 ℃ for 14 h. The recombinant protein was identified as Lb Ⅱ by SDS-PAGE and visible spectrophotometry methods. After the response surface experiment, the titer could reach 7.30 μg/mL in recombinant expression. This study would lay a basis for the subsequent fermentation and production of cowpea hemoglobin in other gene engineering strains.