An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for the determination of dexamethasone (DEX) was developed using soybean peroxidase (SbP) as an enzyme label. A mixture of 3-(10′-phenothiazinyl)-propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) was used as an enhancer of SbP-induced chemiluminescence. Varying the concentrations of the capture antigen (DEX-ovalbumin) and specific anti-DEX antibody, the conditions of the assay were optimized. The values of IC₁₀, IC₅₀ and working range (IC₂₀–IC₈₀) of the CL-ELISA of DEX were 0.02, 0.9, 0.08–9.3 ng mL⁻¹, respectively. It was shown that a pretreatment of cow milk samples by centrifugation and 25% methanol prevented the matrix effect of whole milk. The coefficient of variation (CV) and recovery values from the spiked milk samples estimated by the developed CL-ELISA were in the range of 2.2 to 9.9% and 82 to 142%, respectively.