The objectives were to develop a novel protein conjugate-based ELISA test for whole milk progesterone with a dynamic range capable of fully profiling estrous cycles in the dairy cow and to study effects of whole milk medium on antibody binding to progesterone-protein conjugates. A series of progesterone-4-ovalbumin conjugates with different length intermediate linkers were applied as coating antigens in an ELISA format to determine antibody-binding performance in whole milk. Use of an 18-atom linker gave higher binding than use of a 4- or 11-atom linker at certain conjugate concentrations, but no further increase was observed with increasing linker length. An ELISA constructed with the 18-atom linker conjugate gave a detection limit of 0.089 ng/mL progesterone and correlated well to an established radioimmunoassay procedure (r = 0.94). The assay has the distinct advantages of a wide linear range (0.1 to 100 ng/mL), allowing full profiling of bovine estrous cycles, use of whole milk directly without extraction or prior dilution, and employing more easily purified protein conjugates as coating antigens compared with commercial progesterone-enzyme conjugate for milk ELISA assays.