Abstract: Interleukin-12 (IL-12) is a heterodimeric cytokine composed of p40 and p35 subunits linked by an interchain disulfide bond, and it plays a broad and essential role in the immune system. To characterize the molecular composition and function of IL-12 isoforms in largemouth bass (Micropterus salmoides), we cloned the cDNA sequences of four IL-12 subunits (p35a, p40a, p40b, and p40c) and analyzed their sequences, revealing conserved cysteine residues that are critical for disulfide bond formation. We examined the tissue-specific expression profile of IL-12 subunits in M. salmoides using quantitative real-time PCR (qPCR). Time-series transcriptome analysis was conducted to track expression changes of IL-12 subunit genes in M. salmoides following Nocardia seriolae infection. Two fusion proteins of IL-12 were overexpressed to investigate their functional characteristics. The results indicated that the amino acid sequences of the four IL-12 subunits contain multiple conserved cysteine residues involved in disulfide bonding. Tissue-specific expression profiling via qPCR revealed differential expression patterns of these subunits across six organs (liver, spleen, head kidney, etc.). Time-series transcriptome analysis showed that the p40c gene was persistently suppressed in both the head kidney and spleen, while p40b expression was continuously suppressed in the head kidney and transiently upregulated in the spleen on the second day post-infection before gradually decreasing. Additionally, p35a, p40a, and p40b exhibited divergent expression profiles in the head kidney and spleen. Co-IP assays confirmed interactions between p35a and p40b/p40c. Overexpression of the two fusion proteins p40b-p35a and p40c-p35a in M. salmoides demonstrated that both dimers formed by p40b+p35a and p40c+p35a significantly upregulated IFN-γ expression in the spleen. Our study identifies two functional IL-12 isoforms in M. salmoides, composed of p40b+p35a and p40c+p35a, providing important insights into IL-12-mediated immune responses in teleost fish.