Objective To establish and evaluate a chronic model of atopic dermatitis (AD) induced by 2,4-dinitrochlorobenzene (DNCB) in Brown Norway rats. Methods Brown Norway rats were randomly divided into negative, model, and tacrolimus groups. The ear skin of rats in model and tacrolimus groups were challenged by 0. 5% DNCB at days 1, 3, 7, 9, 12, 14, 16, 19, 21, 23 and 25. Rats in the control group were administered a substrate solution at the same volume at the same time points. Rats in the tacrolimus group were transdermally administered tacrolimus ointment for 25 collected and weighted after 26 days of modeling. Histopathological examination and toluidine blue staining of ear tissue were also performed. The IgE level in ear tissue was detected. Results The ear thickness in model rats was significantly increased after 9 days of challenge by DNCB, while whereas inflammation score was remarkedly improved after 12 days and reached a peak at 16~25 days. The weight of ear tissue and the IgE level in the ear were increased at day 26 of challenge. Pathological changes, such as epidermal thickening, inflammatory cell infiltration, and mast cell infiltration, occurred in the model group. Tacrolimus significantly reversed these changes. Conclusions Brown Norway rats challenged by DNCB develop stable AD-like symptoms, which are appropriate for pharmacological evaluation.