A novel reagent, acetoacetanilide (AAA), was introduced to the determination of formaldehyde based on Hantzsch reaction. A simple and highly sensitive fluorometric method was achieved by using AAA. The main advantages in the use of this reagent are: the reaction is carried out at room temperature without any heating system, the cyclization product based on Hantzsch reaction is soluble in water, and the product can be detected by spectrophotometry and fluorometry. The maximum absorption wavelength of the product occurs at 368 nm, and the maximum excitation and emission wavelengths are found at 370 and 470 nm, respectively. Several important experimental variables of the procedures were examined; particularly, the reaction temperature, reaction time, concentrations of reagents, and pH of the reagent solution were optimized for improving the detecting sensitivity. The calibration graph was linear in the range of 1 × 10 -7 -1 × 10 -6 M or much higher concentrations. The limit of detection (LOD), based on three times of the standard deviation of the reagent blank, was 2.0 × 10 -8 M. The proposed method was applied to the determination of formaldehyde in environmental water samples. Many foreign species commonly existing in water samples did not interfere with the determination of formaldehyde in the proposed method.