Abstract: Objectives: The present study aimed to achieve the efficient and soluble expression of chondroitinase ABC Ⅱ (ChSase ABC Ⅱ) and investigate its enzymatic properties. The study thus laid the groundwork for the application of ChSase ABC Ⅱ in pharma- and nutraceutical production industries. Methods: Based on the optimization of the original sequence of ChSase ABC Ⅱ gene, the recombinant plasmid pET-28a-His-ChSase ABC Ⅱ was constructed and the expression conditions of the recombinant plasmid were optimized. The partial enzymatic properties of His-ChSase ABC Ⅱ were studied after obtaining ChSase ABC Ⅱ fusion protein with a polyhistidine-tag (His-tag) using affinity chromatography. Results: The results showed that the constructed His-ChSase ABC II fusion protein expression system successfully achieved the soluble expression of the protein in Escherichia coli. In the optimal conditions that the expression host was E. coli BL21(DE3) and inducer (isopropyl-β-D-thiogalactoside) concentration was 125 μmol/L, the enzyme activity of fermentation broth reached 7206.83±184.27 IU/L. Furthermore, the purified His-ChSase ABC Ⅱ had an enzyme specific activity of 22.02±0.39 IU/mg protein, with optimal pH and temperature of 7.5 and 40 ℃, respectively. The enzyme was also found to be stable at 30~40 °C with a half-life of over 2 h. Moreover, the His-ChSase ABC II was determined to specifically and effectively decompose chondroitin sulfate, with a K_m value of 10.4±0.8 μmol/L and a K_cat value of 9.4±0.2 s⁻¹. Conclusions: Efficient expression and purification of His-ChSase ABC Ⅱ were achieved using gene optimization and fusion expression strategies in this study. Moreover, the enzymatic properties of the recombinant fusion protein could fundamentally meet the standards essential for its application in the industrial production of pharma- and nutraceutical products.