Phenoloxidase (PO) is a multicopper enzyme, which plays an important role in melanin synthesis. PO is necessary for defense against intruding microorganisms and parasites, and is important in wound healing and cuticle pigmentation. As a specific inhibitor of PO, kojic acid (KA) is commonly used to characterize the functional properties of PO. In this study, we investigated the effect of KA on PO activity during the development of the oriental fruit fly, Bactrocera dorsalis, which is a destructive pest of many horticultural crops. After feeding on a KA-containing artificial diet, the larval and pupal developmental periods were significantly prolonged. In addition, the larvae did not grow to normal size and rates of pupation and emergence were decreased when B. dorsalis larvae had been fed with KA-containing diet for 6 days. Compared to the control, PO activities from whole bodies of B. dorsalis were inhibited, so did in larval cuticles. Consistent with these, kinetic analysis showed that the catalytic capability of PO was significantly reduced. The I₅₀ values of KA for PO activity also indicated that KA was an effective inhibitor of PO in B. dorsalis. Biochemical characterization showed that PO from B. dorsalis had maximum activity at pH 7.5 and 37 °C. These results provide additional understanding of the role of PO in growth of B. dorsalis. As an inhibitor of PO, KA is capable of disrupting the development of this pest insect.