Abstract: The aims of the present study were to assess the effect of exogenous enzymes supplementation on in vitro cultural characteristic of microbes in rumen fermentation of Lake Sheep and to provide scientific references for the application of exogenous enzymes in animal feedstuff. Four levels of exogenous enzymes, including 10, 20, 30, 40 mg/kg, were supplemented into in vitro rumen fermentation cultural system respectively. The rumen microbes were co-cultured with exogenous enzymes, and the gas production （GP）, pH value, NH3-N concentration, MCP concentration and VFA concentration in the cultural fermentation were determined at 6 th ,12 th,18 th, 24 th, 36 th and 48 th h after the start of culture. The results showed that the GP, pH value, NH 3-N concentration, MCP concentration and VFA concentration in the rumen microbes fermentation of Lake Sheep in vitro were effectively increased by supplementation of different levels of exogenous enzymes, and the 10 mg/kg of exogenous enzymes exhibited the best performance.