In order to develop the optimum process of anti-cancer peptides preparation from Ruditapes philippinarum, five proteases were selected to hydrolyze the clams under the same solid-liquid ratio, reaction time, enzyme dosage, optimum temperature and pH conditions. The amino nitrogen content and antitumor activity were measured using formaldehyde potentiometric titration and Cell counting kit-8(CCK-8) methods, respectively, and the correlation between the two indexes was analyzed. Enzymolysis conditions were optimized using single factor experiment and orthogonal design. The results showed that the enzymatic peptides of flavored protease had the highest content of amino nitrogen; whereas the enzymatic peptides of pepsin had the highest antitumor activity. Optimal hydrolysis conditions of pepsin were confirmed as: solid-liquid ratio 1:1(w/w), pH value 2.0, reaction time 1 h, enzyme in solid matter 2 000 U/g, and, enzymolysis temperature 37 ℃. At the dose of 20 mg/mL, the enzymatic peptides that were obtained under the optimal conditions had an inhibition rate of 99.83% on A549. Thus the types of enzymes exert significant influence on the antitumor activity of enzymatic peptides, and there was no direct positive correlation between the content of amino nitrogen and the inhibition rate on A549 in the enzymolysis solution of Ruditapes philippinarum.