Phenoloxidase (PO) is one of the most important factors in the bivalve immune defense system. In the current study, the PO of the scallop Argopecten irradians was purified from hemocytes using linear-gradient native PAGE combined with catechol and Coomassie brilliant blue staining. The results show that purified PO had a molecular mass of 555 kDa in native PAGE and 55 kDa in denatured PAGE. The analysis of the kinetics indicated that the K_m values of PO for L-DOPA, catechol, and hydroquinone were 0.51 mM/L, 0.23 mM/L, and 0.87 mM/L, respectively, which suggests that the PO was a type of p-diphenoloxidase. The PO had optimal activities at 40°C and a pH of 8.0, and its activity was greatly enhanced by Ca² and Mg² , and inhibited by Fe² and Mn² . In addition, the PO activity was inhibited by sodium sulfite, ascorbic acid, sodium diethyldithiocarbamate (DETC), cysteine, citric acid, and ethylenediamine tetraacetic acid disodium (EDTA). However, no inhibition was found when thiourea and sodium azide were used. Based on the inhibition that was induced by EDTA and DETC, we conclude that the PO of A. irradians was a copper-containing metalloenzyme.