The subject research to make the Horse Radish Peroxydase-progesterone (HRP) using for ELISA reaction in order to determine progesterone (P4) concentration. The result after two times of attachment received 2.05 ml composite of solid HRP-P4 (sell-attached HRP-P4). After determining the standard curve, the subject determined the suitable dilution rate of sell-attached HRP-P4 using for reaction ELISA is 1/500000. Using sell-attached HRP-P4 for ELISA reaction to determine concentration P4 in order to diagnose early pregnant with 29 reproductive cross-bred cows F2, F3. 16 cows are diagnosed pregnancy that have P4 concentration from 0.25nglml to 0.28ng/ml in estrus day (day 0), then increase quickly geting 0.88nglml at 71h, 1.83 ng/ml in 2151 and 2.41 ng/ml in day 25th after insemination.Using sell-attached HRP-P4 can be able to replace HRP-P4 import in EIA-P4 Kit in ELISA reaction to determine concentration P4 in order to diagnose the early pregnant of cows.