One of the main enzymes found in papaya is papain, which is used in many different fields. Therefore, in order to use papain must be collected, isolated, and purified from the papaya plants. This study discusses the extraction and purification of papain from papaya latex (PL). Three-Phase Partitioning (TPP) technique was used for extraction and purification. Protease activity, protein content, specific activity, activity recovery, and purification fold of both the intermediate and bottom layers were determined to investigate the variation in these parameters in the separated layers of latex. Then, after extraction of papain enzyme from the latex, the effects of different parameters, including pH, temperature, and papain concentration, on protease activity were tested to optimize papain activity. In this respect, it was found that pH 7, 60 ⁰C and a concentration of 2% were the optimum parameters for protease activity of the papain enzyme. In addition, the antioxidant activity of the extracted PL from papaya fruits was evaluated using total antioxidant activity, reducing power, and DPPH assays. The antioxidant activity, reducing power, and DPPH scavenging activity of PL increased gradually with increasing PL concentration. Furthermore, the total phenolic content (TPC) and total flavonoids content (TFC) of PL were determined to confirm the potential antioxidant activity of PL. It was found that TPC and TFC were 141 µg eq. GA/mL, and 71 µg eq. QE/mL, respectively.