B7-H4, a new member of the B7 family, may participate in the negative regulation of cell-mediated immunity, while aberrant B7-H4 expression is detected in some tumors and it participates in the occurrence and development of the tumors. In this study, we developed one monoclonal antibody (mAb) whose clone No. was 4H8 against the extracellular domains of B7-H4 through immunization of Balb/c mice with the 3T3-mB7-H4 cells that expressed extrinsic B7-H4 stably. And we detected the expression characteristics of B7-H4 in various tumors using 4H8 mAb by enzyme-linked immunosorbent assay (ELISA), immunoprecipitation and immunohistochemistry (IHC) analysis. B7-H4 expression was significantly higher in the tumor tissues derived from uterus, breast, and colon than in their corresponding normal tissues. Further, the B7-H4 expression was related to the stage of the tumors. In contrast, B7-H4 expression did not differ significantly between the tumor tissues derived from the stomach and liver and the normal tissues. Different expression levels of B7-H4 in the tumors indicated that B7-H4 may be involved in tumor formation and development. Specific mAbs against B7-H4 will be useful in studying the role of B7-H4 in tumor pathogenesis and pathological process.