Disturbance in the balance between pro-oxidants and anti-oxidants result oxidative stress in aerobic organisms. However, oxidative stress can be inhibited by enzymatic and non-enzymatic defense mechanisms. Superoxide dismutases (SODs) are well-known scavengers of superoxide radicals, and they protect cells by detoxifying hazardous reactive oxygen species. Here, we have identified and characterized two different SODs, CuZnSOD and MnSOD, from black rockfish (RfCuZnSOD and RfMnSOD, respectively). In silico analysis revealed the well-conserved molecular structures comprising all essential properties of CuZnSOD and MnSOD. Phylogenetic analysis revealed that both RfCuZnSOD and RfMnSOD cladded with their fish counterparts. The recombinant RfSOD proteins demonstrated their potential superoxide scavenging abilities through a xanthine oxidase assay. The optimum temperature and pH conditions for both rRfSODs were 25 °C and pH 8, respectively. Moreover, the potential peroxidation function of rRfCuZnSOD was observed in the presence of HCO3−. The highest peroxidation activity was observed at 100 μg/mL of rRfCuZnSOD using the MTT cell viability assay and flow cytometry. The analogous tissue-specific expression profile indicated ubiquitous expression of both RfCuZnSOD and RfMnSOD in selected tissues of healthy juvenile rockfish. An immune challenge experiment illustrated the altered expression profiles of both RfCuZnSOD and RfMnSOD against lipopolysaccharide, Streptococcus iniae, and polyinosinic-polycytidylic acid (poly I:C). Collectively, these results strengthen the general understanding of the structural and functional characteristics of SODs within the host defense system.