This study evaluated the role of probiotics in enhancing intestinal immunity and mitigating polystyrene nanoplastics (PS-NPs)-induced toxicity in grass carp (Ctenopharyngodon idella). Grass carp were fed probiotics (Bacillus subtilis, Bacillus velezensis, Lactobacillus reuteri, and Lactococcus lactis) for two weeks before being exposed to PS-NPs for five days. Probiotic pretreatment alleviated PS-NPs-induced intestinal damage, with Bacillus velezensis and Lactococcus lactis groups showing milder vacuolation and villus breakage than other groups. Probiotic-treated fish exhibited transient increases in antioxidant enzyme activities (CAT, SOD, MPO) and immune gene expression (IL-6, IL-8, IL-10, IL-1β, TNF-α, and IFN-γ2) shortly after exposure, followed by significant downregulation over time. Higher abundance of the gut dominant phylum Proteobacteria was observed in four probiotic groups exposed to PS-NPs than that in the blank control group. The Clostridium phylum showed a significant decrease in the abundance both in the LRS-PS100 and LLS-PS100 groups, while the abundance of the Thick-walled phylum increased. The Spearman correlation matrix revealed that specific gut microbiota, such as Serratia, Neisseria, and Lactococcus, were significantly associated with enzymatic activities and immune system related genes’ expressions. Probiotic pretreatment enhanced the intestinal immune response of grass carp. However, this enhanced immune response was insufficient to counteract the toxic effects of PS-NPs exposure, particularly in terms of oxidative stress levels and gut microbial diversity. This study offers new insights into the potential of probiotics to combat NPs pollution in aquaculture. It emphasizes the need for further research to explore various probiotic combinations. Future studies should also investigate optimal dosages and durations to effectively mitigate the biological toxicity of NPs pollution.