High-performance liquid chromatography with UV detection using an acetylaceton derivatization was developed in order to determine the presence of formaldehyde (HCHO) in seawater. Several operating and derivatization conditions were examined to analyze the low concentration of HCHO in seawater containing some coexisting species. When 40 (v/v) % methanol was used in the mobile phase, HCHO derivative was detected at nearly 5.4 min. The calibration curve obtained from the peak height for the HCHO derivative was linear, with a good correlation coefficient of 0.99. The relative standard deviation (n=10) of the peak height was 3.4 %. The detection and quantitation limits were 3 μg/L and 11 μg/L, respectively. Although inorganic species have little effect on this method, organic species show an opposite tendency. Using a 2.5 fold concentration of the derivatization reagents in the derivatization reaction, the interference due to the organic species was resolved. This developed method was applied to the determination of HCHO in seawater samples.