The dairy industry traditionally uses aspartic proteases from bovine and microbial sources for cheese processing, but there is interest in alternative enzyme sources to supply the large global demand for rennet. This study investigated the biochemical characterization of a partially purified pepsin of yellowfin tuna with milk-clotting activity (MCA), in addition to evaluating its potential use in the manufacture of fresh cheese through texturometric and organoleptic characterization. The molecular weight of tuna pepsin was 36 kDa. The optimal temperature of MCA was recorded at 60 °C. Caseinolytic activity and MCA of tuna pepsin peaked at pH 6. A high MCA was recorded when 0.018% CaCl₂ was added to the reaction mixture. The electrophoretic profile of casein hydrolyzed by tuna pepsin showed a 14.8 kDa fragment, a molecular weight like that of para-κ-casein. Cheeses made from commercial chymosin and tuna pepsin showed a similar protein content (11% of total wet weight); however, cheese produced with pepsin had a higher fat content. The cohesiveness and chewiness of cheese made with partially purified pepsin were significantly higher than those made with chymosin. There were no differences in the sensory analysis between cheese made with both clotting agents; however, 60% of panelists preferred cheese made with chymosin. Pepsin extracted from yellowfin tuna stomach showed interesting milk-clotting properties with potential biotechnological use, which would contribute to the reusing and revalorization of byproducts derived from the tuna processing industry.