Cynanchum otophyllum Schneid. contains the coagulant protease. The leaves of Cynanchum otophyllum are widely used to produce the milk cake (a traditional cheese) by the minority ethnic region in Yunnan Province. Milk cake made by Cynanchum otophyllum leaves usually forms a soft curd. The object was to extract the active coagulant components based on the leaves of Cynanchum otophyllum and apply the extract into milk cake manufacture. The results showed that the optimal extraction temperature to produce Cynanchum otophyllum coagulant was 4℃, while the optimal extraction liquid-solid ratio was 20mL/g and the optimal extraction solution was the citric acid-phosphate buffer (containing 150mmol/L NaCl, 1mmol/L Cys and EDTA). Compared with the milk cake made by the soaking solution of Cynanchum otophyllum leaves, the yield, protein and fat recovery rate of milk cake made by the extracted coagulant were significantly improved, with the recovery rate for protein and fat of 20.26 and 33.04 percentage points, respectively. Moreover, the milk cake made by extracted coagulant showed a more suitable hardness, higher degree of adhesion and more uniform gel network structure compared with the milk cake made by soaking solution. The study provided a reference for the development of Cynanchum otophyllum coagulant and promoted the industrialization of Yunnan traditional milk cake products.