Firstly, polyphenol oxidase (PPO) was purified from the fruits of Opuntia ficus-indica using Sepharose 4B–L-tyrosine–p-aminobenzoic acid affinity chromatography, and the enzyme was characterized. The PPO was purified 20.59-fold. Thereafter, PPO was performed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The kinetic parameters, optimum pHs, and optimum temperatures were investigated for three substrates. Opuntia ficus-indica PPO’s optimum pH and optimum temperature were 9.0 and 20 °C; 7.5 and 20 °C; and 7.5 and 30 °C, respectively, when using pyrogallol, catechol, and 4-methyl catechol as substrates. For the pyrogallol, catechol, and 4-methyl catechol, the Km, Vmax, and Vmax/Km values were determined as 16.67 mM, 833.33 U/mLmin, and 50 U/mLminmM; 6.33 mM, 126.58 U/mLmin, and 20 U/mLminmM; and 5.38 mM, 107.53 U/mLmin, and 20 U/mLminmM, respectively. As a result, pyrogallol was a more appropriate substrate than catechol and 4-methyl catechol for the PPO from Opuntia ficus-indica.