A sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of 17β-estradiol (17 β-E₂) was developed. Optimisation of two ELISA competition assays, using monoclonal or polyclonal antibodies anti-17 β-estradiol, coupled with the electrochemical detection was firstly performed. The activity of the label enzyme (horseradish peroxidase) was measured electrochemically using 3,3′,5,5′-tetramethylbenzidine as substrate. The use of the polyclonal antibody resulted in a more sensitive assay and the detection limit of the assay was estimated to be 20 pg ml⁻¹. The analytical performances of the method were compared to those obtained using a dissociation enhanced lanthanide fluorescence immunoassay (DELFIA). Although sample extraction is not usually required by DELFIA, both extracted and non extracted samples were assayed. The comparison between the two screening techniques revealed similar results for the extracted samples and showed a comparable precision (RSD%), ranging from 6.2 to 13.4 and from 6.7 to 14.3 for DELFIA and ELISA, respectively. The results obtained by these screening assays were confirmed by liquid chromatography atmospheric pressure chemical ionisation tandem mass spectrometry which is currently used to confirm illegal hormone administration for regulatory purposes. The electrochemical enzyme immunoassay appears suitable as a screening tool for routine analysis of bovine serum estradiol and can be extended to other anabolic hormones using appropriate antibodies.