This study aimed to produce polyclonal anti-body (Abpo) equine chorionic gonadotropin (eCG) in Madura bulls. The Abpo-eCG was an alternative hormone for the superovulation technique and estrus synchronization of ruminant. The isolation of sera eCG derived from local Indonesian pregnant mare sera at 1-3 months age had beed collected from jugular veins, and then purification with charcoal was done by chromatography Sephadex G100. After that, the produce of Abpo eCG have done in two Madura bulls carried out injected with 3,000 IU eCG from local Indonesian pregnant mare sera 3 times at 10 day intervals. Furthermore, Abpo eCG was isolated from serum taken from the jugular vein of bull, then extraction was done by adding absolute charcoal and ethanol and centrifuged at 3000 rpm for 15 minutes at 4oC. Next step, the identification of Abpo-eCG that producted from Madura bull by SDS-PAGE 12% method, Western Blot and Elisa indirect to find out the highest level at the 7th week of 408.50 mlU /mL. Then purified by CM Sephadex G-100 coloumn chromatography technique. The final product was made as a Frozen Dry dosage form.