Osteopontin (OPN) and 2,-fucosyllactose (2,-FL) in human milk are important bioactive components that regulate intestinal immunity and barrier function in infants. To investigate the synergistic effects and mechanisms of OPN and 2,-FL combinations on intestinal barrier function, a soybean meal-induced zebrafish larval enteritis model was established. The study intervened with OPN and 2,-FL monomers and combinations at different ratios (1:18~1:1, m/m) and measured the number of intestinal neutrophils and goblet cells, expression levels of inflammation- and barrier-related genes, and protein expression levels of key cytokines. The results demonstrated that 1:9~1:3 (m/m) OPN:2,-FL ratio significantly reduced the number of neutrophils to 13.4 and 13.2 per larva and increased the number of goblet cells to a level close to that in the normal group (95.5 per larva). Meanwhile, the gene expression levels of claudin-1/3, zonula occludens-1, and mucin 5.1 were upregulated by 3~4 fold. Additionally, the gene expression levels of toll-like receptor 4 and nuclear factor-κB were reduced by 47.33% and 57.12%, respectively. The immune homeostasis of the organism was improved by regulating the inflammatory cytokine protein expression levels. In summary, the combination of OPN and 2,-FL significantly synergistically improve intestinal barrier function in zebrafish larvae with enteritis, providing a theoretical basis for the optimization of infant formula design.