This study was carried out to evaluate the uncertainty in the analysis of total alkaloids and reducing sugar content in flue-cured tobacco. The sources of uncertainty associated with the analysis of total alkaloids and reducing sugar were the weighing of sample, the preparation of extracting solution, the addition of extracting solution into the sample, the preparation of standard solution, the precision of calibration curve for standard solution, the reproducibility of analysis, and the determination of water content in tobacco, etc. For the calculating uncertainties, Type A of uncertainty was evaluated by the statistical analysis of a series of observation, and Type B by the information based on supplier’s catalogue and/or certificated of calibration. It was shown that the main source of uncertainty was caused by the calibration curve of standard solution, the reproducibility of analysis, the volume measurement of 1$mell$, and the purity of nicotine reference material in the preparation of standard solution. The uncertainty in the addition of extracting solution, the sample weighing, the volume measurement of 100$mell$, and the determination of water content of tobacco contributed relatively little to the overall uncertainty. The expanded uncertainty of total alkaloids and reducing sugar in flue-cured tobacco at 95% level of confidence was $pm$0.12% and $pm$0.54%, respectively.