To assess the uncertainty in the determination of cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) in industrial cannabis using High-performance liquid chromatography (HPLC), according to 'Evaluation and expression of uncertainty in measurement' (JF1059.1-2012) and 'Guidelines for the evaluation of uncertainty in chemical analysis' (CNAS-GL006-2019), a mathematical model of uncertainty evaluation for determination of CBD and THC in industrial hemp by HPLC was established. The sources of uncertainty were analyzed and the components of uncertainty were calculated, then the synthetic uncertainty and extended uncertainty were evaluated. The results showed that the main factors of uncertainty in the process of CBD were as follows: preparation of standard solution > fitting of standard working curve > repeated times > extraction > weighing of samples. In the process of THC detection, the main factors of uncertainty were as follows: standard working curve fitting > times of repetition > preparation of standard solution > extraction > weighing of samples. The average value of CBD was 0.166 g/100 g, the expanded uncertainty was 0.0312 g/100 g, the result was X = (0.166 ± 0.0312) g/100 g, the inclusion factor k = 2. The average value of THC was 0.0522 g/100 g, the expanded uncertainty was 0.00344 g/100 g, X = (0.0522 ± 0.00344) g/100 g, k = 2. This method is suitable for the evaluation of uncertainty of CBD and THC under similar conditions.