【Aim】 Nanopore sequencing technology, a single-molecule real-time sequencing approach, plays a crucial role in rapid clinical diagnosis and microbiological detection. Using Tephritidae as a crucial example of quarantine pests, the applicability of nanopore sequencing technology was assessed for insect quarantine identification, offering a new assessment approach. 【Method】 We employed Sanger sequencing technology and nanopore sequencing to obtain the DNA barcodes of 14 Tephritidae species previously identified based on morphology. Each sequencing result was cross-referenced in the NCBI and BOLD databases to confirm species accuracy. We then compared the accuracy of sequences obtained through these two distinct sequencing technologies for each species. 【Result】 Nanopore sequencing generated 181-megabyte bases in just 44 min across 14 samples, with an average of 11280 reads per sample and individual read accuracy of 92.10%-94.53 %. After sequence correction for consistency, the results of nanopore sequencing were consistent with those of Sanger sequencing, and the sequencing analysis findings were consistent with the outcome of morphological identification. 【Conclusion】 Our study demonstrates the full applicability of nanopore sequencing technology to quarantine the identification of Tephritidae using the experimental process and data analysis methods outlined here. The sequencing results proved to be both accurate and efficient. Additionally, the experimental protocol provided in this study is suitable for species identification based on amplicon sequencing, fulfilling the requirements for high-throughput and accurate identification of large-scale samples.