Objective To evaluate the feasibility of DNA barcoding applied to the identification of flies in Fujian province, China, and to set up the nucleic acid sequence database for flies in Fujian. Methods Sixty-four fly specimens belonging to 19 species, 13 genera, and 4 families were collected from Fujian. Genomic DNA was extracted from the flies. Mitochondrial COⅠ gene was amplified by consensus primer PCR and sequenced. Sequence alignment and evolutionary divergence were performed, and a neighbor-joining phylogenetic tree was constructed based on these sequences. Results Intraspecific divergence ranged between 0-3.32%; interspecific divergence ranged between 5.26%-18.30%, except for Lucilia cuprina and Lucilia sericata, which had only 0.69% divergence from each other. The result of phylogenetic tree showed that sequences from the same species gathered closely with each other. The reliability of Bootstrap test was all above 96%. Conclusion DNA barcoding can be effectively used to identify some flies in Fujian province. It can be used as an efficient method supplementary to morphological identification.