Ebola virus (EBOV) causes uncommon but dramatic outbreaks in remote regions of Africa, where diagnostic facilities are limited. In order to develop diagnostic tests, which can be handled and distributed easily, monoclonal antibodies (mAbs) to EBOV, species Zaire, were produced from mice immunized with inactivated viral particles. Nine stable hybridoma cell lines were obtained producing specific mAbs directed against the viral structural protein VP40. These mAbs were characterized by enzyme-linked immunosorbent, immunoblot and immunofluorescence assays. Subsequently, an antigen capture enzyme-linked immunosorbent assay was established, which detects VP40 of all known species of EBOV. This assay could detect viral material in spiked human serum that has been sodium dodecylsulfate-inactivated. The established enzyme-linked immunosorbent assay therefore has the ability to become a very useful tool for obtaining an accurate diagnosis in the field, limiting the risk of laboratory infections.