In order to study vitellogenesis and its endocrine regulation, we used hybridoma techniques and developed four monoclonal antibodies to Pteromalus puparum soluble yolk proteins, named as PpVt mAb1, PpVt mAb2, PpVt mAb3, and PpVt mAb4, respectively. The four antibodies, each with a IgG1 heavy chain and a κ light chain, had high specificity and affinity not only to the ovarian vitellin (Vt), but also to female hemolymph vitellogenin (Vg). However, they showed no immunological reaction with the male substances. The indirect double antibody sandwich enzyme linked immunosorbent assay (ELISA) was regarded as the most sensitive and accurate to measure Vg/Vt in the wasps, compared with other three methods of ELISA, namely, direct, indirect and double antibody sandwich ELISA. With this ELISA method it is possible to detect the Vg/Vt titer in a single wasp with the sensitivity of 20 ng/mL. The Western blot analysis indicated that the Vg synthesis initiated just after eclosion, and peaked to high levels within 12 and 36 h after eclosion. 