The Chinese longsnout catfish (Leiocassis longirostris) is an economically important freshwater fish in China; males grow faster than the females, suggesting that all-male production is economically desirable in aquaculture. However, their sex determination, sex differentiation genes, and the related regulatory mechanisms remain unknown. In this study, transcriptome sequencing of the ovaries and testes of Chinese longsnout catfish was performed using an Illumina high-throughput sequencing platform. We obtained 358048456 raw reads and 356168472 clean reads from the transcriptome. In total, 24661 genes were annotated, of which 23708 genes were aligned with the reference genome. Gene expression patterns in the ovaries and testes were compared. The results showed that there were 10872 differentially expressed genes (DEGs) between the ovary and testis, including 9375 up-regulated genes and 1497 down-regulated genes. 21 functional DEGs were selected for RT-qPCR analysis and the results confirmed the reliability of the transcriptome analysis. The DEGs were annotated using the Nr, KEGG, and GO databases, where they were enriched in 58 GO functional annotation components and 338 KEGG secondary branching metabolic systems. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the differentially expressed genes were significantly enriched in 16 pathways related to sex determination and differentiation and gonadal development, including the Wnt signaling, TGF-β signaling, MAPK signaling, ovarian steroid signaling, and GnRH signaling pathways. Through functional annotation, 71 important sex-related candidate genes were screened, including 50 highly expressed genes (such as dmrt1, cyp17a1, samd7, wnt6, and wt1) and 21 highly expressed ovarian genes (such as foxl2, gdf9, zp3, zp1, figla, and bmp15). Protein–protein interaction (PPI) network analysis revealed that the testis specificity gene dmrt1 interacts with the testis-biased DEGs (sox9, wt1, cyp17a1, rspo1, gata4, and hsd3b) and the ovary-biased DEGs (foxl2, figla, and bmp1), suggesting that dmrt1 as a sex-determining gene can play a role by inducing the testisbiased DEGs and inhibiting the ovary-biased DEGs during testicular development. In this study, the differentially expressed genes related to sex determination and differentiation of Chinese longsnout catfish were identified and the signaling pathways involved in gonadal development in both males and females were revealed. These findings provide important data for future research on the mechanisms of sex determination and differentiation of Chinese longsnout catfish and thus provide theoretical support for all-male seed production in Chinese longsnout catfish.