Polyphenol oxidase (PPO) plays a critical role in decrement of shrimp quality. To obtain active PPO and elucidate its enzymatic properties, PPO from Litopenaeus vannamei (Lv-PPO) was cloned, expressed in E. coli and purified by affinity column chromatography. The Lv-PPO gene was 2076 bp in length encoding 691 amino acids. The recombinant Lv-PPO (rLv-PPO) with a molecular mass of ∼85.0 kDa was successfully expressed and its sequence was verified by LC-MS/MS. rLv-PPO was biologically active with an optimal temperature of 40℃ and an optimal pH of 6.0. Metal ions Cu2+ and Zn2+ altered the activity of rLv-PPO by influencing its secondary and tertiary structures. rLv-PPO showed catalytic activity towards l-Dopa and catechol. A specific polyclonal antibody against rLv-PPO was prepared. Western blot analysis revealed that PPO levels were highest in hemolymph, followed by telson, carapace, and eyestalk. Expression of rLv-PPO will assist future studies on the mechanism in shrimp melanosis.