A degranulation model was established using mouse bone marrow-derived mast cell (BMMC) to investigate the anti-allergic activity of milk exosomes (M-Exo). Casein was removed from cow’s milk using a rennet-assisted method, and then M-Exo was isolated from the milk by ultracentrifugation and characterized. Different concentrations of M-Exo were used to treat sensitized BMMCs, and the release rate of activated β-hexosaminidase (β-HEX) and cytokine secretion were measured. The results showed that the prepared M-Exo had a typical tea tray-like bilayer membrane structure, with particle size ranging from 30 to 200 nm. M-Exo contained exosome marker proteins CD81, tumor susceptibility gene 101 (TSG101) and heatshockprotein 70 (HSP70). It was further found that M-Exo could be taken up by BMMCs. In the degranulation model, it was confirmed that M-Exo could significantly inhibit the release rate of β-HEX and downregulate the expression of tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), and IL-13 in a dose-dependent manner, indicating that M-Exo has anti-allergic activity.