Abstract: Sheep blood as the research material and the yield of heme was measure indicate. Orthogonal array design generated based on single-factor experiments was employed to optimize conditions. The extraction factors such as the amount of enzyme, pH, react temperature and react time were investigated. The results showed that the optimal amount of enzyme was 8000U/g, pH8.5, react temperature was 55℃, react time was4 h, under these optimal conditions, the yield of heme was 85.3%, that was equivalent to 3.93 g heme of 1L sheep blood. This method had higher safety than others, it was suitable for industrialized production.