The oriental fruit fly, Bactrocera dorsalis, is a major pest that infects fruits and agricultural products worldwide. The latest resistance monitoring of B. dorsalis from mainland China has identified high levels of resistance to insecticides. In this study, the biochemical and molecular characteristics of acetylcholinesterase (AChE) in four field populations of B. dorsalis are investigated. Among the four populations, the DG population and its purified AChE were found to be the least susceptible to malathion and five inhibitors, whereas the KM population and its purified AChE were the most susceptible. The highest catalytic activity of purified AChE was found for the KM population, and the catalytic activity of the DG population was the lowest. Among developmental stages, the AChE purified from larvae was found to be the most insusceptible to inhibitors, but its catalytic activity was the highest. Sequence analysis of the cDNA encoding AChE showed that some residue differences existed. However, no significant differences in expression levels of the AChE gene among populations and developmental stages were detected. The results suggest that the decrease in susceptibility of B. dorsalis was mainly caused by decrease in AChE activity, and they provide a broad view on the relation between AChE and resistance.