This study was conducted to determined, effects of addition of garlic oil (GO) 0 (control), 100, 200, 400, 800, 1200 and 1600 mg/L rumen fluid (RF) by in vitro gas production technique, on the true organic matter digestibility (TOMD), organic matter digestibility (OMD), neutral detergent fiber digestibility (NDFD), metabolizable energy (ME) compound and rumen fermentation parameters, carbon dioxide (CO2) and methane (CH4) gas production of the sainfoin (Onobrychis sativa L.) hay. In vitro gas production technique was used to determine the effects of different doses of GO on rumen fermentation, OMD and metabolic energy (ME) level. GKMS and NDFS contents of sainfoin hay were determined by Daisy incubator technique. The addition of GO significantly decreased the in vitro gas production, OMD, NDFD, ME, total volatile fatty acids content (VFA), acetic acid (AA), propionic acid (PA), butyric acid (BA) and the other volatile fatty acids levels of sainfoin hay (P<0.01). In addition, the addition of GO at different doses to the rumen fluid also reduced the production of CH4 and CO2 production (P <0.01). As a result, in vitro gas production, rumen fermentation, nutrient digestion, methane and carbon dioxide production on the most adverse effect garlic oil dose was found to be 1600 mg / L RF. It was concluded that it would be appropriate to use low doses (400 mg / L RF) because of the high GO doses that affect rumen fermentation and the digestion of feed.