MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate various biological processes including sexual dimorphism. The oriental fruit fly Bactrocera dorsalis is one of the most destructive agricultural insect pests in many Asian countries. However, no miRNAs have been identified from the separate sex and gonads to elucidate sex gonad differentiation in B. dorsalis. In this study, we constructed four small RNA libraries from whole body of females, males (except ovaries and testes) and ovaries, testes of B. dorsalis for deep sequencing. The data analysis revealed 183 known and 120 novel miRNAs from these libraries. 18 female-biased and 16 male-biased miRNAs that may be involved in sexual differentiation were found by comparing the miRNA expression profiles in the four libraries. Using a bioinformatic approach, we predicted doublesex (dsx) as a target gene of the female-biased miR-989-3p which is considered as the key switch gene in the sex determination of tephritid insects. This study reveals the first miRNA profile related to the sex differentiation and gives a first insight into sex differences in miRNA expression of B. dorsalis which could facilitate studies of the reproductive organ specific roles of miRNAs.