The invention provides an in-vitro application of peptide which is used for regulating the cow rumen fermentation and a detection method thereof. The method can exactly detect the influence of peptide on the rumen fermentation parameters such as gas production, pH value, ammoniacal nitrogen value, volatile fatty acid and MCP index which are importance indexes for the rumen fermentation. The detailed situation of fermentation in the active rumen can be directly known by the detection; furthermore, the influence of the peptide on the microorganism enzyme activity can be exactly measured; amylase, proteolytic enzyme and carboxymethyl cellulose activity are main enzyme activity indexes in the rumen; the change of microorganism in the active rumen can be directly known by the detection of the three enzymes. The method has the advantages of convenience, being easy for learning, simple and convenient operation, high repeatability, exact numerical value and strong simulation verisimilitude, can effectively determine the approximate consistency which is suitable for peptide in the rumen of cow, and can give out scientific basis and theoretical guidance for deeper theoretic research, preparing the daily ration for the cow more exactly, reducing the waste of protein feeds and improving the quality of the milk products in the future.