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Fatty acids (FAs) play a vital physiological role in lipid metabolism, which is reported as potential diagnostic biomarker for various diseases. Thus, it is urgent to develop a credible method that can profile FA metabolism with a holistic view. Here, a targeted strategy to screen FAs was developed by parallel labeling with d 0 /d 6 -dansylhydrazine (d 0 /d 6 -DnsHz) and using ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UPLC–MS/MS) in data-dependent MS/MS (ddMS2) mode. The simple and mild derivatization procedure within 3 h allowed for a significant improvement in sensitivity. Additionally, the characteristic product ions introduced by the derivatization reagent assist to identify the unknown FA species. A quantitation method was established by multiple reaction monitoring (MRM) and the d 6 -DnsHz tagged standards for each analyte were used as internal standards to overcome the matrix effects. By applying the method to determine FA levels in plasma collected from the esophageal squamous cell carcinoma (ESCC) patients and healthy controls, 65 FA metabolites were characterized and six FAs were found to be altered by the invasion of tumors. The parallel derivatization strategy provides insights into the identification of unknown FAs and paves a new way for targeted metabolomics. Also, this novel method is a powerful tool for characterization and quantification of FAs in biological samples, which shows a great potential application in clinical diagnosis and investigation of disease mechanisms. [Display omitted] • A novel parallel derivatization strategy was developed to identify FAs in biosample. • Dansylhydrazine-based derivatization enhanced ionization efficiency of FAs. • The isotope-labeled standard provides one-to-one internal standard for FA analysis. • Six FAs were screened out as potential diagnostic biomarkers of ESCC. [ABSTRACT FROM AUTHOR]