As the worldwide elderly population has grown, the incidence and prevalence of Alzheimer's disease (AD) has steadily increased. The differential lifelong exposure of populations to dietary fatty acids has raised concerns about the potential links between cognitive impairments and nutrition. However, few studies have addressed the levels of free fatty acids (FFAs) in AD patient serum. In this study, gas chromatography-mass spectrometry (GC-MS) was used to determine the levels of 15 serum FFAs in 31 AD patients and 33 healthy controls. The optimized methodology entailed the formation of methyl esters using 10% v/v H2SO4/CH3OH at 62°C for 2 hours. The linear range was 0.55-300 μg/mL, the range of recovery was 85.1-104.3%, and the detection limit was 0.03-0.08 μg/mL. Several FFAs in the AD patient significantly decreased when compared to the control, including three saturated fatty acids (C14:0, C16:0, and C18:0) and six unsaturated fatty acids (C16:1, C18:1, C18:2, γ-C18:3, C20:2, and C22:6). The serum level of C18:3 was significantly higher in the AD patients. The FFA profiles of the AD patients differed significantly from those of controls. The method effectively determined the FFA levels and could facilitate future studies regarding the relationship between AD and the metabolism of FFAs.