A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the human receptor activator of nuclear factor κB (RANKL)-osteoprotegerin (OPG) complex was developed by utilizing a monoclonal antibody that recognizes human soluble RANKL as an immobilized capture component and biotinylated human OPG polyclonal antibody. We could quantify the RANKL-OPG complex level (detection limit: 100 pg/mL). Employing this assay system, we demonstrated that the RANKL-OPG complex was constitutively present in the conditioned medium of human osteoblastic sarcoma U2OS, although the complex was not detectable in other human osteoblastic sarcoma cell line, MG-63, HOS, and SaOS-2.