Tephritid fruit flies (Diptera: Tephritidae) include serious agricultural insect pests in the world. Besides causing severe damage to fruits and vegetables, this kind of pests could enter countries or regions with international trade easily. Strict trade quarantine measures are imposed in many countries or regions in order to prevent their introduction and spread. Thus accurate and rapid identification is regarded as an essential component of plant quarantine. Traditional expert systems for assistant identification of agricultural insect pests are based on their morphological characteristics. Compared with the morphological identification, however, molecular identification has more advantages especially for the identification of the immature samples which are intercepted more frequently. Among the molecular identification methods, DNA barcoding is very effective and has been selected by the taxonomists in recent 5 years. In view of the above, a network expert system based on the DNA barcode, Tephritid Barcode Identification System (TBIS) was developed with ASP.NET and C# to improve the molecular identification of fruit fly pests in China. The system was supported by Microsoft SQL server 2008 database. Three functions were provided such as molecular identification based on DNA barcode, information browse and inquiry. DNA sequence similarity alignment dynamic programming algorithm served as the inference mechanism. Molecular identification knowledge was obtained from the public database on the Internet and Plant Quarantine Laboratory of China Agricultural University, which contained about 400 COI sequences of nearly 150 species of fruit flies. Moreover, detailed information such as morphological description and pictures of adult, hosts, and geographical distribution are presented in this system. Mixed with molecular, morphological and distributional data, the system can be used as an identification tool both for quarantine technicians and for educational purposes in China.