Excessive use of plastics in daily life is causing plastic pollution in aquatic environment and threatening the aquatic life. Therefore, research on the plastic pollution in aquatic environment is crucial to understand its impact and develop effective solution for safeguarding aquatic life and ecosystem. The current study investigated the effects of water borne polystyrene nanoparticles (PS-NPs) on hemato-immunological indices, serum metabolic enzymes, gills, and liver antioxidant parameters, plasma cortisol level and histopathological changes in liver and gill tissues of the widely distributed fish Hypophthalmichthys molitrix. The fingerlings of H. molitrix were exposed to different concentrations (T1–0.5, T2–1.0, and T3–2.0 mg/L) of PS-NPs respectively for 15 days consecutively. Our results indicated the dose dependent negative effects of PS-NPs on the physiology and histopathology of H. molitrix. Immuno-hematological indices showed significant increase in WBCs count, phagocytic activity, and lysozyme activity, while decreased RBC count, Hct%, Hb level, total proteins, IgM, and respiratory burst activity were observed. The levels of antioxidant enzymes like SOD, CAT and POD showed the decreasing trends while metabolic enzymes (AST, ALT, ALP and LDH), LPO, ROS activities and relative expressions of SOD1, CAT, HIF1-α and HSP-70 genes increased with increased concentrations of PS-NPs. Moreover, blood glucose and cortisol levels also showed significant increasing trends with dose dependent manner. Histopathological examination indicated moderate to severe changes in the gills and liver tissues of the group treated with 2.0 mg/L of PS-NPs. Overall, the results showed the deleterious effects of PS-NPs on physiology, immunity, metabolism, and gene expressions of H. molitrix. It is concluded that particulate plastic pollution has deleterious effects on filter feeding fish, which might affect human health through food chain and particulate chemical toxicity.