Abstract: The monitoring and research of volatile oxygenated organic pollutants, including aldehydes and ketones, have garnered widespread attention. Given the existing shortage of formaldehyde-2,4-dinitrophenylhydrazone standard substances for high-performance liquid chromatography (HPLC)-based formaldehyde measurement, this study embarks on exploring the synthesis method of formaldehyde hydrazone through the derivatization reaction of formaldehyde and 2,4-dinitrophenylhydrazine. It also delves into the qualitative and quantitative analyses of the resulting product. By optimizing reaction parameters such as reaction time, the acidity of the reaction solution, and the number of extractions, the study has identified optimal conditions for the derivatization reaction: a reaction time of 30 minutes, a reaction solution pH of 2, and 6 extraction iterations. Infrared spectroscopy and gas chromatography-mass spectrometry were employed to qualitatively confirm the derivatives, while HPLC was used for quantitative analysis, with a subsequent evaluation of measurement uncertainty. The results indicated that the synthesized formaldehyde-2,4-dinitrophenylhydrazone aligns well with the NIST Standard Spectrum Library, and could achieve a purity of up to 98% and an uncertainty of less than 1%, making it a promising candidate for standard substance formulation. Additionally, the established HPLC method has been proven capable of accurate formaldehyde-2,4-dinitrophenylhydrazone measurements. This study will offer vital insights for the preparation, valuation, and development of standard substances like formaldehyde-2,4-dinitrophenylhydrazone.