Aim: In forensic medicine, measurement of ethanol levels is important in lawsuits filed against individuals. Each measurement result has a certain amount of uncertainty. The objective of this study is to determine uncertainty of ethanol measurement using the alcohol dehydrogenase method, and also to evaluate sources of variability and their contributions to uncertainty. Material and Method: Repeatability, calibrator uncertainty, and stability uncertainty were determined for ethanol measurement. After their standard uncertainties were calculated, the squares of each value were summed and the combined standard uncertainty value was found with the square roots of that value. The expanded uncertainty value was obtained by multiplying the combined standard uncertainty value by a coverage factor (k=2; confidence interval, 95%). Results: The uncertainty of measurement for ethanol was found to be ±8.5% (confidence interval, 95%). The result at threshold level (50 mg/dL) was reported as 50±4.3 mg/dL (confidence interval, 95%). Discussion: Measurement uncertainty should be estimated to ensure accuracy and reliability of the results in an accredited laboratory performing alcohol measurement. Sources of error that might affect the results during the preanalytical, analytical, and post-analytical phases should be identified and the uncertainty value should be calculated.