The present study was conducted on proteases biosynthesis by Penicillium griseoroseum and its application to cheese formation. Thirty fungal strains of Penicillium griseoroseum were screened for the production of protease using solid substrate fermentation. Results indicated that Penicillium griseoroseum HUV-21 gave the best results for the biosynthesis of acidic protease. Different cultural conditions were employed to enhance protease production in the fermented mash. The enzyme synthesis was maximum (61.85 U g-1), when soybean meal was used as substrate. The optimum temperature, incubation period and depth of substrate were found to be 30 C, 72 h and 20 mm, respectively. It was concluded that protease concentration of 6.0 U g-1 was the best for milk clotting.