A commercially available trypsin-chymotrypsin mixture was covalently immobilized onto modified polyvinyl chloride (PVC) microspheres, which were activated by the subsequent treatment of PVC microspheres with ethylenediamine and glutaraldehyde. The immobilized mixed protease was characterized by FT-IR and SEM analyses. Immobilization conditions were optimized by Box-Behnken design and the response surface method. The activity of the immobilized mixed protease prepared under optimal conditions (pH 6.6, 23 °C, 2 h) reached 1341 U/g. Compared with the free form, the immobilized enzyme possesses a slightly higher optimal pH value and a wider pH-activity profile, superior thermal stability, and a higher Km value. Reusability of the immobilized mixed protease indicated that >70% of the original activity was retained after having been recycled six times.