Sex of fish is determined by both genetic substance and environment. The studies on the relationship between the sex and sex-related genes expression can not only reveal the mechanisms of sex determination, but also provide the theoretical basis to control the sex differentiation in artificial propagation in the fish. Catfish (Clarias fuscus) is a common freshwater fish in China. The Dmrt1 cDNA sequences were isolated from the testes of C. fuscus by RT-PCR and SMART RACE. Structure and function of the predicted DMRT1 protein were analyzed by bioinformatics. Expressions of Dmrt1 were examined using semi-quantitative RT-PCR in ten tissues (testes, ovaries, muscles, intestine, livers, heart, head kidney, gills, brains and eyes) and the testes from stageⅡ to stageⅤ. Results showed that the Dmrt1 complete cDNA was 1417 bp in length, and the 5 un-translated region (5-UTR) and the 3 un-translated region (3-UTR) were 35 bp and 516 bp, respectively. The open reading frame (ORF) was 864 bp, and a sequence of predicted 287 amino acids residues was encoded. The C. fuscus DMRT1 shared a higher amino acid identity of 83.3% to 96.1% with three Siluriformes fishes such as C. gariepinus, C. batrachus and Pelteobagrus fulvidraco. DMRT1 protein coded by Dmrt1 gene located mainly in nucleus, and worked as an unstable hydrophobic protein. A highly conserved DM domain was found in the DMRT1 protein, which was a common characteristic in DMRT clusters. The C. fuscus shared a higher DM domain identity of 100% with the above-mentioned Siluriformes fishes, 91.9% to 97.3% with Danio rerio, Oryzias latipes and Oncorhynchus mykiss, and over 80% with chicken, rat, pig and human. In addition, the DM domain of C. fuscus had a typical C2H2C4 zinc finger structure, which bound with the downstream DNA and control the gene transcription. Expression of Dmrt1 was found only in the testes, but not in ovaries, muscles, intestine, liver, heart, head kidney, gills, brain and eyes, which suggested that Dmrt1 was a testis-specific gene in C. fuscus. The expression of Dmrt1 in testes stageⅡ(spermatogenesis) was significantly higher compared with the testes from stageⅢ to stageⅤ(P0.05), which indicated that Dmrt1 was related to sex determination, spermatogenesis and testes development in male C. fuscus.