Vitellogenin (VTG), a phospholipoglycoprotein precursor of egg yolk is synthesized and secreted in the liver in response to circulating estrogens in female fish. Thus, the presence of VTG in male fish is a useful biomarker to identify estrogenic activity of natural or anthropogenic substances in sewage effluents. We report the purification of carp (Cyprinus carpio) and perch (Perca fluviatilis) VTG with the subsequent development and characterization of a specific ELISA for VTG measurement. VTG was purified by combination of ion exchange chromatography and size exclusion chromatography. The purified proteins were used as antigen for antibody production, as standard and tracer in the assay. Carp VTG was stable and showed a characteristic double band after Western blotting of the purified protein and in serum samples, respectively. In perch samples, several bands with lower molecular weight were present and appeared to be degradation products of VTG. The development of the carp VTG ELISA led to a sensitive and valid test system with inter-assay coefficients of variation between 3.0 and 12.3%. In contrast to carp, the described ELISA for perch VTG showed a much higher inter-assay variation up to 24%, possibly attributable to fast protein degradation. In conclusion, the described two-step chromatography is a simple purification method for VTG. Immunological and electrophoretical test systems are valid methods to determine VTG in some species like carp, but for other species like perch in which VTG is not stable, these methods are not applicable.