Abstract: In order to improve the accuracy of test results, the uncertainty in the determination of benzoic acid in tobacco casing by HPLC was systematically calculated according to JJF 1059-1999 Evaluation and Expression of Uncertainty in Measurement. The results indicated that: 1) calibration curve and recovery of benzoic acid were the main factors influencing the uncertainty;2) the combined uncertainty was 0.23 mg/kg and the expanded uncertainty was 0.46 mg/kg (level of confidence 95%, k=2) for the tobacco casing contained 6.98 mg/kg of benzoic acid. It was concluded that the uncertainty in determination of benzoic acid in tobacco casing by HPLC could be reduced by using high purity standard, high precision containers, and strictly controlling of preparation procedures.