Otitis media with effusion (OME) is a disease with a complicated pathogenesis that remains unclear. Previous studies have shown that allergy is one of the pathogenic factors involved in OME. However, the regulatory mechanism of allergy in OME is unknown. This study evaluated the T helper cell 1 (Th1)/Th2 polarization tendency in the middle ear microenvironment. To investigate the differentiation of Th cells in the middle ear of a rat model of OME, 20 Sprague-Dawley rats were randomly divided into an OME group (Exp group) and a control group (Con group). The levels of interferon (IFN)-gamma and IL-4 in the tympanic lavage fluid were evaluated by ELISA. Additionally, the levels of IFN-gamma and IL-4 in the middle ear mucosa and bone marrow cavity were examined by immunohistochemistry. Compared to those in the Con group, the levels of IL-4 and ratio of Th2/Th1 (IL-4/IFN-gamma) in the tympanic lavage fluid, middle ear mucosa, and bone marrow of the Exp group were increased significantly, whereas there was no significant difference in IFN-gamma levels between groups. The expression of IL-4 in the middle ear highly increased following allergy stimulation, whereas the expression of IFN-gamma was decreased. A Th1/Th2 immune response imbalance results in a Th2 response in the middle ear microenvironment of an allergic OME rat model.