Abstract: Microbial sources of bioagents are coinciding with the attempts to find cheap alternative substrates for high production. Rennet is the golden element in the dairy industry, particularly cheese making. Commonly, the use of statistical models to optimize culture medium components and conditions has increased in present-day biotechnology, due to its ready applicability and aptness. The present study adopted statistical approach for Milk Clotting Enzyme (MCE) production by Mucor mucedo KP736529 which was isolated, selected and molecularly identified, as the most potent active strain in MCE biosynthesis. Screening of various medium constituents using the statistical Plackett-Burman design showed glucose and KH2PO4 as the significant factors affecting MCE biosynthesis. Optimizing MCE production by the central composite design led to 6.12 fold increase in MCE production, compared with initial activity. Domiati cheese was manufactured using the M. mucedo KP736529 enzyme (E-cheese) or using commercial calf rennet (C-cheese) as control. During two months under pickling conditions, the yield and chemical properties of E-cheese were better than those of C-cheese. The fungal enzyme (MCE) showed proteolytic activity higher than calf rennet with the absence of taste defects such as bitter taste. Organoleptically, E-cheese scored the highest points.