A method was developed for the measurement of acetaldehyde in human blood by high-performance liquid chromatography (HPLC). The method was based on a pre-column reaction; a fluorescent substance was formed by a coupling reaction between 2mol of cyclohexan-1, 3-dione and 1 mol of acetaldehyde with ammonium acetate. The coupling compound was analyzed by HPLC and the concentration of acetaldehyde was obtained from the calibration curve drawn from the results using standard solutions. The determination was sensitive and reproducible with a range of 0.2-10 μM and a precision (coefficient of variation) of 2.43%.