TBT residual in water had become a noticeable ecological problem for aquatic ecosystems. The river pufferfish (Takifugu obscurus) is a kind of an anadromous fish species and widely distributed in the East China Sea and the Yellow Sea. Because of the water contamination, the pufferfish wild resource had a sudden decline in recent years. Therefore, the study on the response of pufferfish to the TBT exposure may contribute to reveal toxic injury mechanism of T. obscurus under TBT exposure. In this study, the transcriptional library of T. obscurus liver and gill was constructed and sequenced by an improved Illumina HiseqX10 high-throughput sequencing platform under different concentrations of TBT acute stress. The blood cell numbers distinctly decreased after TBT exposure, showing the adverse effects of TBT invasion and self-adjusting ability of the pufferfish. The production of reactive oxygen species increased, demonstrating the oxidation resistance of T. obscurus when exposed to TBT. The obtained data were compared with the genome data of Takifugu rubripes and transcriptional resource database. On this basis, gene function annotation, analysis and classification were carried out by bioinformatics method, and differential genes related to toxic injury function were screened out. Meanwhile, new toxic related genes and related signal pathways were sought to provide new theoretical guidance for the pathogenesis of T. obscurus exposed to TBT. This study not only enriched the transcriptome data of T. obscurus under environmental stress, but also provided a new research method for the response mechanism of T. obscurus under the stimulation of environmental factors.