Objective To establish a mouse model of allergic conjunctivitis by comparison of different antigens, routes of administration, and ages of mice. We compared their immune response status and screened for optimal sensitization conditions to provide new ideas and method to establish mouse models of allergic conjunctivitis. Methods Sixty-five SPF Balb / c mice were selected to establish allergic conjunctivitis models using different antigens ( ovalbumin, ragweed pollen, house dust mite ), administration routes ( gastric administration, aerosol inhalation, subcutaneous injection) and ages ( 1, 2, 3 and 8 weeks). Results The expression levels of interleukin-17 ( IL-17) and C-C motif chemokine ligand 5 ( CCL5) mRNA were increased in the ovalbumin, house dust mite and ragweed pollen groups, especially the later, compared with the negative control group. Compared with the negative control group, the expression of IL-17 mRNA was increased in the subcutaneous injection, gavage, inhalation and positive control groups. The expression of CCL5 mRNA in the gavage group was close to that of the negative control group, while its expression level in the subcutaneous injection group was closer to that of the positive control group. Expression levels of CCL5 and IL-17 mRNA were similar in 1 - and 2-week-old mice, but significantly lower than those in 3 - and 8-week-old mice. Conclusions Ragweed pollen provided better sensitization in the preparation of an allergic conjunctivitis mouse model. Subcutaneous injection of antigen achieved satisfactory result in the allergic conjunctivitis mouse model. Although there were no obvious eye symptoms, the whole body and local areas were sensitized. The immune response of 3-week-old Balb / c mice was close to that of adult Balb / c mice.