The Polyphenol oxidase (PPO) leads to the enzymatic browning of fruits and vegetables and needs to be prevented in food browning and quality. The present study aimed to investigate the use of natural extracts and chemical inhibitors to prevent browning of the PPO of blackberries. Purification, characterization, and kinetics of PPO of blackberry parameters for five substrates, namely, pyrocatechol, 4-methylcatechol, Pyrogallol, Gallic acid, and tyrosine, were described. The results showed that the DEAE-Sepharose and Superdex G-200 purification methods, which achieved electrophoretic purity, increased PPO activity by 556 fold. Purification with Sephadex GE-200 and SDS-PAGE reveals two PPO isoenzymes with an apparent molecular weight of 22 kD and 70 kD. The optimum pH and temperature values indicated were 6.6 and 25°C, respectively. The PPO showed variable affinity towards o-dihydroxy phenolic substrates with catecholase activity but without any activity observed with phenol, a monohydroxy substrate, and it was very effective towards pyrocatechol, pyrogallol, and 4-methyl catechol. The results revealed that inhibition of the PPO using both synthetic inhibitors and natural extracts was the most effective method. Quercetin and ascorbic acid showed higher inhibition with the lowest Ki values. Fresh onion (Allium cepa) and wild Arbutus unedo extract were able to inhibit the blackberry PPO activity up to 50% and 60%, respectively. Therefore, the use of natural extracts from Arbutus unedo L as anti-browning agents on the blackberry PPO may provide new insight to overcome the enzymatic browning.