[Aim] The fruit fly Bactrocera tuberculata is a phytosanitary pest in China that can be severely affect a wide range of potential hosts. It is morphologically similar to its relatives and is commonly identified by examining adult external morphological characteristics, which is relatively time-consuming. This study aimed to develop a more rapid method of identifying B. tuberculata from any instar using molecular identification. [Method] Implementing species-specific mitochondrial DNA (mtDNA) cytochrome oxidase subunit I (SS-COⅠ) primer technology, B. tuberculate was used as the positive control, and the remaining 19 types were used as negative controls. Genomic DNA templates were extracted, PCR amplification was performed, and the products were analyzed via electrophoresis. The COⅠ mtDNA sequence was chosen, the homologous series were checked, and B. tuberculata SS identification primers LFJ400 and LJR564 were designed. [Result] Only B. tuberculata could clearly amplify the approximately 165 bp single band, whereas the rest produced no band. [Conclusion] The SS-COⅠ primers designed with high specificity could rapidly and accurately identify B. tuberculata and were suitable for various insect instars. This method is of great practical significance for the prompt quarantine, identification, and clearing through customs of the pests of imported fruits and vegetables.