Ribonucleases (Rnases)2 and Rnase3 belong to the ribonuclease A (RnaseA) superfamily. Apart from their role in molecular evolutionary and functional biological studies, these genes have also been studied in the context of defense against pathogen infection in mammals. However, expression patterns, structures and response to bacterial infection of the two genes in blunt snout bream (Megalobrama amblycephala) remain unknown. In this study, we identified multiple copies of Rnase2 (six) and Rnase3 (three) in the M. amblycephala genome. The nine genes all possess characteristics typical of the RnaseA superfamily. No expression was detected in the early developmental stages, while a weak expression was observed at 120 and 140 h post-fertilization (hpf) for Rnase2b, Rnase2c, Rnase2e and Rnase3a, suggesting that only three copies of Rnase2 and one of Rnase3 are expressed. Interestingly, only Rnase2e was up-regulated in the kidney of M. amblycephala after Aeromonas hydrophila infection, while Rnase3a was significantly up-regulated in liver, gut and blood after the infection. We conclude that the paralogs of Rnase3 are more susceptible to A. hydrophila infection than Rnase2. These results indicate that different Rnase2 and Rnase3 paralogs suggest a role in the innate immune response of M. amblycephala to bacterial infection.