The present work was aimed to develop fresh cheese using the crude extract from calotropis procera leaves and thereby performed evaluating milk‐clotting activity and acute toxicity of crude extract. The extraction of coagulant was performed at 30, 45, and 60℃ using 2, 6, and 10 g of dried calotropis procera leaves, respectively. The highest yield of crude enzyme extract was acquired with 2 g and 30℃, and results were varied significantly (p < .05). The highest cheese yield and fastest clotting time were 17.89 kg cheese/100kg milk and 14:50 min acquired with 10 g C. procera powder at 60℃ extraction temperature, respectively. E. coli, total bacterial count, yeast, and mold microbial load detection were observed below the limit established by codex alimentarius. This study recommends that use of C. procera crude enzyme leaves extract can be used as alternate milk coagulant for production of fresh cheeses. Soft cheeses manufactured with calotropin enzyme as milk coagulant revealed no adverse effects of toxicity tested on albino rates to the highest dose.