Abstract: In order to obtain sex-differential expression genes and explore the sex determination mechanism of Trionyx sinensis, we sequenced the transcriptome of testis and ovary of T. sinensis by RNA-seq and obtained clean data of 21 494 396 400 bp and 24 605 142 600 bp, respectively. A total of 10 145 differentially expressed genes were obtained, of which 4 034 were up-regulated in ovaries and 6 111 were down-regulated in ovaries. Through GO, KEGG and protein interaction analysis, 117 genes with sex-different expression were obtained in the study of the sex-difference of soft-shuttles. The sex-related genes such as Foxl2, Sox3, Sox7, Bmp4, Csf1, Wnk2, Mtmr4, Gsc, Mr2f2, Fgf9, Tgfβ2, Tgfβ3, Rara, Smd2, Smd3 and Mad5 were detected. The sex-related genes with high expression in males were Dmrt1, Amh, Sox30, Cct4, Cct5, Cldn11, Crem, Dhcr24, Dnaii, Dynll1, Gabarap, Herc4, Herpud2, Hexb, Hook1, Hormad1 and Smc4. At the same time Bmp4 regulated the expression of Foxl2 through Smad signaling pathway and Foxl2 gene was a marker gene for female physiological sex of T. sinensis.