Epithelial-derived thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that is mainly produced by epithelial cells. It has been shown to play a key role in the development of Th2-type allergic inflammation. Commercial antibodies available against TSLP can only be used in Western blot assay, which further limits investigation of its function. Here we efficiently generated a panel of anti-mouse TSLP monoclonal antibodies in rats with DNA priming-protein boosting strategy. Overall, four MAb strains (4B12, 4E11, 5D7, and 6H10) were obtained and their characterizations were identified. The MAbs can specifically bind to TSLP according to the ELISA and FACS assays. It was found that they recognized distinct epitopes. They are useful in detecting TSLP expression in the tissue by immunoblotting and in the cytoplasm by intracellular staining assay. Thus, these antibodies will be valuable tools for studying TSLP biological functions.