As global extreme heat events become more frequent, aquaculture faces significant challenges due to prolonged high summer temperatures, which lead to elevated water temperatures. Investigating the physiological and biochemical responses of fish to thermal stress, as well as breeding heat-resistant varieties, are essential strategies for addressing these challenges. Thamnaconus septentrionalis is an important aquaculture species in southern China. When water temperatures exceed 30 °C during the summer, this species experiences reduced feeding, growth stagnation, and increased mortality rates. To elucidate the physiological and biochemical response mechanisms of T. septentrionalis under thermal stress, this study established a high-temperature group (30 °C) and a control group (20 °C) for a 48-h thermal stress experiment. Tissue samples were collected from the experimental fish at 0, 12, 24, and 48 h post-stress. The effects of acute thermal stress on gill tissue structure, serum biochemical indicators, liver antioxidant capacity, and liver transcriptomics were explored. The results revealed that the gill tissues of the high-temperature group exhibited slight bending of the gill filaments, terminal swelling, and cellular vacuolization, along with a tendency for adjacent gill filaments to fuse. The liver antioxidant capacity and serum biochemical indicators in the high-temperature group were significantly elevated compared to the control group (P < 0.05). Transcriptomic sequencing identified 5536 differentially expressed genes (P < 0.05), with 2639 genes upregulated and 2897 downregulated. Gene Ontology (GO) enrichment analysis indicated that differentially expressed genes were primarily associated with metabolic processes and redox reactions. KEGG pathway analysis showed significant enrichment of genes in pathways related to amino acid metabolism, carbon metabolism, and glycolysis/gluconeogenesis, with notable downregulation observed in the high-temperature group. This study provides valuable theoretical insights into the mechanisms underlying the response of T. septentrionalis to high-temperature stress, contributing to the understanding of aquaculture resilience in the face of climate change.