Abstract: As the raw material, hyaluronidase was isolated from bovine liver, the optimistic conditions was investigated. Plackett -Burman experiment, steepest ascent experiment and Central composite design were performed. The results showed that the optimistic isolation parameter were pH4.69 of acetate buffer, 0.22mol/L NaCl and extraction time 10.82h. Under these conditions, the specific activity of hyaluronidase from bovine liver reached 33.27U/g. By ammonia sulfate salting-out and Sephacryl S-100 preliminary purification, the specific activity reached 452.81U/g.