Abstract: The spectrophotometric determination method about L-carnitine in organs of yak was optimized and the content of L-carnitine in the yak organs was analyzed in this paper. Based on the single factor experiment, the response surface methodology was used to select the enzyme reaction time, pH of buffer system and enzyme reaction temperature. These three was shown as variables, and absorbance as response value. The optimum conditions were as follows:the enzyme reaction temperature was 37℃, pH was 7.6 and enzyme reaction time was 16 minutes. Under this condition, the absorbance of L-carnitine was 0.321. The measured value of yak heart, liver, lung and kidney was analyzed under the optimum condition. And the result was shown that the highest L-carnitine yield of 28.35mg/kg was achieved from liver, then was lung of 22.75mg/kg and heart of 14.61mg/kg, kidney had the lowest content of 5.22mg/kg.