Interleukin-12 (IL-12) is an important cytokine that connects the innate and adaptive immune systems and has been shown to play an increasingly important role in immune and inflammatory responses. The study of IL-12 in fish is mainly focused on cloning and expression, but the study of IL-12 in Megalobrama amblycephala, one of the economically important freshwater fish, has not been reported yet. Therefore, in this study, four IL-12 subunits in M. amblycephala were cloned, sequenced and phylogenetically analyzed; the expressions of IL-12 in healthy tissues and after infection by Aeromonas hydrophila in vivo and stimulated by LPS in vitro were detected; and the function of the recombinant proteins of IL-12Bb and IL-12BC was verified. The results showed that the open reading frame (ORF) sequences of IL-12A、IL-12Ba、IL-12Bb and IL-12Bc were 588, 993, 939 and 837 bp, respectively. Quantitative real-time PCR (RT-qPCR) results showed that the four IL-12 subunits were expressed in all the 10 different healthy tissues tested, but their expression patterns were different. After intraperitoneal injection of A. hydrophila, the M. amblycephala IL-12 subunits were significantly induced in the immune-related tissues. After LPS stimulation, the four IL-12 subunits were rapidly induced in the head-kidney lymphocytes (HKL) of M. amblycephala. Furthermore, the recombinant IL-12Bb and IL-12Bc proteins were obtained by Escherichia coli prokaryotic expression system, and then were used to stimulate HKL. The results showed that the expressions of TNF-α, IL-1β and IFN-γ in the stimulated HKL were significantly up-regulated, indicating that the obtained recombinant proteins had biological activity. Finally, the antimicrobial activity of the obtained protein was tested, and the results showed that the recombinant IL-12Bb and IL-12Bc monomers had obvious inhibitory effects on A. hydrophila, E. coli and Staphylococcus aureus. The above results of this study indicate that IL-12 plays an important role in the process of regulating immune response in M. amblycephala, which will provide theoretical basis for further study of IL-12 in fish.