Objective The uncertainty evaluation of caffeine in beverages was measured by HPLC method, and the uncertainty was analyzed for providing a good reference for improving determination method and finding the key steps in determination. Methods In accordance with JJF-1059-1999 method, finding the factors which impacts the uncertainty evaluation in determination process, and the components of the uncertainty were evaluated and combined. Results The measurement uncertainty of caffeine in beverages by HPLC methods were evaluated(2.2 μg/mL). Conclusion Uncertainty in determination process was evaluated with a standard curve for quantity, which possesses guiding significance for finding the key steps in the job and improving determination quality in the future.