Transmissible gastroenteritis virus (TGEV) is a member of coronaviruses. The viral spike (S) protein mediates the interaction between TGEV and its susceptible cells. Here, we expressed a truncated gene encoding the N terminal half of TGEV S gene (designated S1 gene) in a prokaryotic system. The resulting S1 protein was used to immunize BALB/c mice followed by the generation of a monoclonal antibody (MAb). A generated MAb (7F9) was identified by ELISA and the chromosome number of the hybridoma cell was analyzed. The immunoreactivity of the MAb to TGEV S protein was confirmed by Western blot analysis. Moreover, immunofluorescence assays showed that the MAb is able to detect cell infection by TGEV. The MAb achieved in this study can be used as a specific diagnostic reagent for detecting TGEV S protein.