M AXIMUM hyaluronidase production by Streptomyces roseofulvus S10 (LC314796) was attained when it was cultured in submerged fermentation process under favorable conditions, pH 5 at 40ºC for 6 days. Hyaluronidase was purified to its homogeneity by 9.2 fold with molecular weight of 97kDa under denaturing SDS- PAGE. Mg+2 exerted highly stimulatory effect on S. roseofulvus S10 hyaluronidase activity and was significantly reduced in presence of Mn⁺², Zn⁺², and EDTA. Optimum reaction was attained at pH 9 and the pH stability of enzyme ranged between 9-10 at 35°C. To protect the intrinsic activity and half-time of hyaluronidase, several carriers and immobilization of hyaluronidase were investigated. The immobilized enzyme had higher thermal stability than free one with Tm values; 46.1°C and 24.7°C, respectively. Maximum affinity of free and immobilized hyaluronidase was for hyaluronic acid followed by bovine albumin. Free enzyme had a high catalytic affinity of hyaluronic acid compared with immobilized enzyme. Our results demonstrated that S. roseofulvus S10 hyaluronidase was highly stable to pH and high temperature. These properties of long-term stability facilitate its wide range of applications.