Abstract: In order to evaluate the uncertainty in the determination of total alkaloids in flue-cured tobacco with continuous flow method,the cause-effect relationship diagram in the determination was revised and a new mathematical model for measurement uncertainty assessment was set up by analyzing the whole measurement process. The sources of measurement uncertainty,sample concentration and measurement repeatability of autoanalyzer were determined. The results showed that: the measurement uncertainty mainly come from the moisture content in sample. The standard uncertainty of measurement results was 0.05%,and the expanded uncertainty (α=0.05,k=2) was 0.10%.