The rosaceous branch borer, Ospheranteria coerulescens, is an important pest of rosaceous trees. This insect feeds on the twigs and branches of living trees and causes their death. The characterization of the insect phenoloxidase (PO) is of interest when doing comparative investigations, and so that we may be able to understand its biochemical properties. When designing new methods of insect control such as the use of PO inhibitors, an understanding of the biochemical properties is fundamental. In this study, PO from hemolymph of the rosaceous branch borer was purified using ammonium sulfate precipitation, gel-filtration, and ion-exchange chromatography. The biochemical properties were characterized using l-dihydroxyphenylalanine (L-DOPA) as the specific substrate. The apparent molecular weights of the three isoforms of PO were determined by SDS-PAGE to be 85.23, 79.45, and 66.06 kDa. Optimal pH for PO activity was pH 8, and the optimal temperature was 45°C. Phenoloxidase lost less than 50% of its relative activity after a 60 min incubation at the optimal temperature. The effects of ions and chemical materials such as K+, Ba2+, Zn2+ and EDTA on PO showed that PO activity was strongly inhibited by Zn2+. The Michaelis constant (Km) and maximum velocity (V max) were 88.61 mM and 0.14 μmol/min, respectively. The inhibitory effects of kojic acid, 4-hexylresorsinol, and quercetin on PO were determined, and the IC 50s (inhibitory concentration) were estimated as 23.31 for kojic acid, 35.75 for 4-hexylresorcinol, and 60.8 μM for quercetin. The inhibitory potency of kojic acid was 1.54 times higher than that of 4-hexylresorcinol and 2.58 times higher than that of quercetin. Phenoloxidase was effectively inhibited by 4-hexylresorcinol, and the inhibition type was competitive. The inhibition types of PO by kojic acid and quercetin were found to be mixed.