Compared to other organophosphate-resistant and -susceptible (S) lines of Bactrocera dorsalis, the carboxylesterase (CBE) BdE5 in the naled-resistant (nal-r) line has been found to possess remarkable quantitative elevation. Our study attempts to identify the role of BdE5 in naled resistance, and we discovered several points of interest. Firstly, activity staining on native PAGE revealed that the percentage of flies with intensive BdE5 bands in the nal-r line was substantially higher than in the S line, indicating that the BdE5 band correlates with naled susceptibility. Secondly, in vitro and in vivo inhibition assays showed that BdE5 was inhibited by naled in both lines; under diagnostic doses of naled, the overall extent of inhibition on CBEs was much greater in the S line than in the nal-r line. Thirdly, NanoLC-nanoESi-MS/MS analysis used the NCBI database to identify and annotate BdE5 as an esterase FE4-like (XP_011200445.1) in B. dorsalis. Fourthly, rapid amplification of cDNA ends was used to obtain the 2012-bp full-length BdE5 cDNA, which contained an open reading frame of 1770bp and encoded a putative protein of 590 amino acid residues. Phylogenetic analysis revealed that BdE5 is a secreted β-esterase (E clade) closely related to CG6414 (NP_570089), a CBE in Drosophila melanogaster. Finally, our relative quantification real-time PCR data showed a significant elevation in transcript levels of the BdE5 gene in nal-r line. Our results confirmed that BdE5 is correlated with naled resistance and provides further understanding about the identification and molecular characteristics of BdE5 in B. dorsalis.