Papaya (Carica papaya) is one of the most widely known and economically important plantings in both tropical and subtropical countries of the world that used as a potential enzyme and nutrients supplement in the food industry. Papain is one of the most important proteinase enzymes in papaya fruit which can breakdowns proteins and has many applications and benefits in food, pharmaceutical, and leather industries. The main aim of this study was to extract and purify papain enzyme from papaya leaf by using disk mill grinding extraction technique, identification and determination of the ash and moisture content of the papain enzyme. Purification of the extracted papain was done salt precipitation method by using ammonium sulfate. Papain enzyme was identified by using powder skim milk protein which was coagulated when incubated at 25oc, 37oc, and 49oc for 30 minutes and at various PH and substrate level. The optimum temperature, pH and substrate level of papain activity was 37oc,4.7and 20ml respectively. The ash content of papain was 9.200393 % after furnace with a temperature of 800oc for 45 minutes and the moisture content of the enzyme was also 15.007767 % under the oven with a temperature of + 105ocfor 24 hrs. From this study, papaya leaves contain the papain enzyme. Therefore, there is a need for further study on different techniques of extraction and purification, and determination of protein content of the papain enzyme.