The invention belongs to the field of fish sex determination in the field of aquaculture, and particularly relates to a specific DNA fragment for sex determination of Mastacembelus armatus and application thereof. According to the invention, on the basis of the 2b-RAD-seq technology, a genome walking technology is utilized, and a male sex specificity marker of the Mastacembelus armatus is developed; the specific DNA fragment is shown as SEQ ID No. 1 or SEQ ID No. 2. Directed at the specific sequence, the sex of the Mastacembelus armatus can be identified by utilizing conventional PCR amplification and common agarose electrophoresis, and accuracy reaches 100% according to verification results. Compared with previous anatomical detection or genital swelling observation, the technology provided by the invention has the advantages of accuracy, easiness, rapidness, little damage to fish bodies and the like, and the specific DNA fragment provides helps for sexual control breeding of the Mastacembelus armatus, protection of wild resources and development of the breeding industry.