Abstract: Used the internal standard method to quantitative analysis of triglyceride content in mixed glycerides, applying the triglyceride separated from mixed glycerides by Silica gel column chromatography to obtain the high-purity products of triglyceride, which is to be the triglyceride standard. Mixed glycerides separated by TLC, eluted and colored, and then showing photometric values by spectrophotometry at 700±1nm wavelength instruct the content of triglyceride. The average recovery was 99.8%. This method was specific accurate, but precision slightly lower. Study on obtain the high-purity triglyceride standard, which showed a good linear relationship within the range of 10-100μ g/ml. Under insuring about 5 min coloring time conditions, detection results were stability in 1.5h. The limits of detection for the method valued were 0.55 μ g/ml, quantitative detection lower limit of 1.83μ g/ml. This method provides a simple and quick way to determinate of triglyceride content in the mixed fatty acid, through several experiments can reduce the random errors and improve precision.