Obscure puffer, Takifugu obscurus, is an important aquaculture species in China, but the disease problem of this species seriously affects its production and causes huge economic losses. In order to reveal the molecular mechanism of disease resistance, polyinosinic-polycytidylic acid [poly(I:C)] was used to stimulate obscure puffer. At 0, 12, and 48 h (named To0, To12, and To48) after poly(I:C) challenge, the kidneys from obscure puffer were collected for transcriptome sequencing. A total of 54,816 transcripts was generated. Pairwise comparison of the sequencing libraries of tissue samples at these three time points revealed that the number of differentially expressed genes (DEGs) at To12 vs To0, To48 vs To0, and To48 vs To12 were 2039, 776, and 2579, respectively. Gene Ontology (GO) function classification analysis revealed that some DEGs were annotated to GO items for membrane, biological process, molecular function, and metabolic process. Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis of DEGs demonstrated that they mainly presented in immune-related pathways, such as Toll-like receptor signaling pathway, Retinoic acid-inducible gene-I-like receptor signaling pathway and NOD-like receptor signaling pathway. Then, eight genes were randomly selected from immune-related genes for real-time quantitative reverse transcription PCR verification (RT-qPCR), and 22 key immune DEGs were used to construct network functions. This study has obtained a large number of information resources about the transcriptome of obscure puffer, which can provide references for further research on the anti-virus response of obscure puffer.