This study was aimed to extract milk-clotting enzyme from sunflower seeds and to determine its potentiality for manufacturing white soft cheese from cows and goats milk. The seeds were blended and extracted using two types of buffers and milk-clotting and proteolytic activities were evaluated. The enzyme was partially purified using ammonium sulfate fractionation techniques. Results indicated that sunflower seeds extracted with 5% NaCl in 50 mmol/L acetate buffer, pH 5.0, had the highest milk-clotting activity (MCA) and lowest coagulation time compared to that extracted with only acetate buffer (pH 5.0). Ammonium sulfate at 30–50% saturation purified the enzyme to 4.3 folds with MCA of 241.0 U/mL and final enzyme yield of 10.9%. The partially purified enzyme was characterized by SDS–PAGE that showed two bands with molecular weight of 120 and 62 kDa. When compared with other plant enzymes, the partially purified sunflower enzyme was found to have higher milk-clotting activity and lower proteolytic activity. Also, both milk sources and enzyme types significantly affected the cheese yield and curd formation time. The cheese made from cow milk using sunflower enzyme had higher yield compared to that obtained using commercial rennet, whereas the opposite was observed when using goat milk.