Ambrosia artemisiifolia (Amb a) contains many allergens. Allergic conjunctivitis caused by Ambrosia artemisiifolia and its related allergen-specific immunotherapy (AIT) are seldom studied at present. poly(DL-lactide-co-glycolide)-polyethylene glycol (PLGA-PEG) is a very good nano-carrier, which has been applied in the medical field. In this context, we studied the immunotherapy effect and potential mechanism of recombinant Amb a 1 (rAmb a 1)-loaded PLGA-PEG nanoparticles. A mouse allergic conjunctivitis model was established with Ambrosia artemisiifolia crude extract, and the nanoparticles were used for AIT through direct observation of conjunctival tissue, degranulation of mast cells in conjunctival tissue, serum-specific antibodies, cytokines and other assessment models. The treatment of nanoparticles enhanced the secretion of T-helper 1 (Th1) cytokine Interferon-gama (IFN-γ) and the production of immunoglobulin G (IgG)2a (IgG2a), inhibited the secretion of T-helper 2 (Th2) cytokine Interleukin (IL)-13 and IL-4 and the level of IgE. Especially, degranulation of mast cells and expression of mast cell protease-1 (MCP-1) in conjunctival tissue was reduced significantly. In this study, we proved that the nanoparticles prepared by rAmb a 1 and PLGA-PEG have an immunotherapy effect on allergic conjunctivitis in mice.