The purpose of this study was to preliminarily investigate the role of sox9 gene in gonad development and sex differentiation in Takifugu obscurus. The full-length cDNA sequence of sox9 gene in Takifugu obscurus is successfully cloned through the design of degenerate primer amplification, RACE and Real-time PCR technology, and subsequently its corresponding bioinformatics characteristics and the level of tissue and expression were also analyzed. The results showed that the full-length of Takifugu obscurus sox9a gene is 1 248 bp (NCBI accession number:MH218818), including a 684 bp ORF, encoding 227 amino acids; the 5'UTR is 297 bp and the 3'UTR is 267 bp. The full-length of Takifugu obscurus sox9b gene is 1 941 bp (NCBI accession number:MH218819), including a 1 470 bp ORF, encoding 489 amino acids; the 5'UTR is 306 bp and the 3'UTR is 165 bp. The results of homology and phylogenetic analysis showed that the two sox9 sequences shared the highest homology and closest relationship with Takifugu rubripes. The results of multiple amino acid sequence alignment showed that the HMG box domains of the two Sox9 amino acid sequences were highly conserved in mammals and fish. Quantitative fluorescence PCR analysis showed that the two sox9 genes are ubiquitous in all tissues of female and male fish, and they were most highly expressed in the hypothalamus of female fish, a little in the testis, and very little in the ovary. Overall, except for a few tissues, the expression of two sox9 genes in tissues of females was generally higher than that of males. The purpose of this study was to understand the genetic characteristics and physiological functions of sox9 in Takifugu obscurus, and to explore the molecular regulation mechanism of sex differentiation and gonadal development.