Twenty monoclonal antibodies (MAbs) to bovine follicle-stimulating hormone (bFSH) were generated by using the hybridoma technique. Twelve of the MAbs have been shown to bind specifically to bFSH and seven of them were purified from the respective ascitic fluids with the methods of caprylic acid precipitation and hydroxylapatite column chromatography. The purity of the MAbs was in the range of 86-95%. Three of the purified MAbs have been coupled to horseradish peroxidase (HRP). By using a matrix cross-matching procedure, two MAbs reacted with discrete antigenic determinants are selected for a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The designed ELISA procedure could be performed within 60 min in a two-stage incubation and has a minimum sensitivity of 1ngFSH/ml. For serum samples, its mean intra- and inter-assay coefficients of variation were 3.4% and 7.3% respectively. The DAS-ELISA can be used to establish a complete FSH profile in the field of in vitro diagnosis.