According to the detection method of free gossypol in vegetable oil，this paper establishes a mathematical model of uncertainty evaluation for determination of free gossypol in vegetable oil . The main sources of uncertaintyin measurement arising from procedure of testing were analyzed and evaluated. The evaluation results showed that the fitting of standard curve, the performance of liquid chromatograph and the repeatability of sample pretreatment made great contribution to the uncertainty of free gossypol detection results. When the content of free gossypol in edible oil was 48 mg/kg, the expanded uncertainty was 5 mg/kg (k=2).