A direct competitive enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (MAb) was diagnosed with progesterone (P) level of human serum. In high concentrations and large amounts of displacer effect, this monoclonal antibody (MAb) can retain biological activity so that it can be specially combined with progesterone. Under conditions of the existing displaced agent, monoclonal antibody 11F8(3)H5 can maintain high specificity and affinity and can specifically bind progesterone in serum. Progesterone ELISA standard curve was calculated according to the following formula: Logit(y) = -1.358Log(x) + 0.4961, r = 0.9944. The serum progesterone values obtained by this method correlated well with those obtained by chemiluminescent immunoassay (CLIA): the correlative equation was y = 0.7804x + 0.7600, r = 0.9126.