A chymotrypsin-like serine protease was purified from Euphorbia rigida plant latex using 50% acetone precipitation. Enzyme activity was determined using protease substrates and inhibitors. The purified enzyme preparate showed the highest activity for the chromogenic substrate SucAAPFpNA, indicating a chymotrypsin-like activity. The partially purified enzyme was observed as a single band on sodium dodecyl sulfate poliacrylamide gel electrophoresis (SDS-PAGE) and its relative molecular weight was estimated as 66.4 kDa. The pH optima of the enzyme was found to be at pH 8 and the enzyme was stable over a wide range of pH. The thermal optima of the enzyme was found to be at 60 °C and the enzyme was stable up to 60 °C after 1 hour of preincubation. Proteolytic activity was strongly inhibited by a serine protease-specific inhibitor PMSF. HgCl₂ inhibited 51% of chymotrypsin activity. Overall, this enzyme may be considered as a strong candidate for various applications in the food and biotechnological industries due to its stability under harsh conditions.