An improved gas chromatography with mass spectrometry procedure was developed to highlight the esterified fatty acids in 100 μL blood of dengue fever patients in the early febrile phase versus healthy volunteers. 24 adult patients and 24 healthy volunteers were included in this study. The recoveries of targeted esterified fatty acids content were in the range of 92.10-101.00% using methanol/dichloromethane (2:1, v/v) as the extraction solvent. An efficient chromatographic separation of targeted 17 esterified fatty acid methyl esters was obtained. The limits of detection and quantification were within the range of 16-131 and 53-430 ng/mL, respectively. The relative standard deviation of intraday and interday precision values ranged from 0.4 to 5.0%. The statistical data treatment showed a significant decrease of the content of four saturated fatty acids, C14:0, C15:0, C16:0, and C18:0 (P value < 0.05), and also showed a decrease of the content of eight unsaturated fatty acids, C16:1, C18:3n6, C18:2n6, C18:1n9, C20:3n3, C20:4n6, C20:2, and C22:6n3 (P value < 0.05) in dengue fever patients. Moreover, the amount of three omega-6 fatty acids including C18:3n6, C18:2n6, and C20:4n6 was dramatically decreased in the blood of dengue fever patients to a limit of 50 ± 10%.