Abstract: In this research, dried soymilk residue and spent culture substrate of Flammlina velutip were mixed at the ratio of 1∶1(w/w) as the culture medium for Mucor pusillus to generate milk-clotting enzyme (rennet). The process of preparing crude milk-clotting enzyme powder was investigated. The procedure was divided in 7 steps including solid-state fermentation in Erlemeyer flask, water extraction, flocculation with Al2(SO4)3, microfiltration to remove fungi, ultrafiltration to concentrate enzyme, ethanol precipitation under room temperature, vacuum drying at 40℃. Results showed that the temperature for inactivation of rennet was around 52℃ in water solution, while lowered it to 35℃ in ethanol solution. The ethanol concentration for the best effect in rennet precipitation was 70%, which recovered rennet activity of 77.24%. The final product obtained was in crude enzyme powder with its purity in rennet for 65.8% and the recovering efficiency of filtrated enzyme for 69.1%. The biotransformation of rennet in crude enzyme was 4 975 SU per gram of culture medium. Comparing conventional procedure in low-temperature precipitation and freeze-drying, results indicated that the milk-clotting enzyme of Mucor pusillus could be recovered in certain extent of rennet activity even with room-temperature precipitation and 40℃ vacuum drying.