AbstractMicropterus salmoides rhabdovirus (MSRV) is an significant pathogen that causes high mortality and related economic losses in bass aquaculture. There is no effective or approved therapy to date. In this study, we evaluated the anti‐MSRV effects of 22 quinoline derivatives in grass carp ovary (GCO) cells. Among these compounds, 8‐hydroxyquinoline exhibited valid inhibition in decreasing MSRV nucleoprotein gene expression levels of 99.3% with a half‐maximal inhibitory concentrations (IC50) value of 4.66 μM at 48 h. Moreover, 8‐hydroxyquinoline significantly enhanced a protective effect in GCO cells by reducing the cytopathic effect (CPE). By comparing the anti‐MSRV activity of 22 quinoline derivatives, we found that 8‐hydroxyquinoline possessed the efficient active site of 8‐hydroxyl and inhibited MSRV infection in vitro. For in vivo studies, 8‐hydroxyquinoline via intraperitoneal injection exhibited an antiviral effect in MSRV‐infected largemouth bass by substantially enhancing the survival rate by 15.0%. Importantly, the viral loads in the infected largemouth bass notably reduced in the spleen on the third days post‐infection. Overall, 8‐hydroxyquinoline was considered to be an efficient agent against MSRV in aquaculture.