Carica papaya leaf extract obtained by a simple and chemical-free method was first analyzed for levels of protein, polyphenols, proteolytic activity and milk clotting activity, and then evaluated in fresh goat’s cheese production as an alternative to calf rennet. Milk clotting essays, cheese yields, protein fractions and sensory evaluations were determined considering animal rennet as reference. The highest cheese yield was obtained with 0.2% dose of fresh papaya leaf extract. Compared to calf rennet, papaya leaf extract remained active even at 95 °C. After 12 months of cold storage, the clotting ability of liquid extract decreased significantly, whereas the lyophilized extract maintained its full clotting potential. Cheese samples made with papaya leaf extract showed lower levels of casein but higher levels of pre-α-caseins and water-soluble nitrogen than those made with calf rennet. Sensory results proved that the use of papaya leaf extract in fresh goat’s cheese production had no negative impact on color, consistency or taste, but on the contrary, it improved cheese’s odor which was remarkably appreciated by panelists. This natural product could therefore be a good alternative to conventional animal rennet and could be used to attribute specificity to traditional fresh goat’s cheese.