The experiments reported in this research communication aimed to compare the serum nonesterified fatty acid (NEFA) composition in ketotic cows and healthy cows during the perinatal period. NEFAs play significant roles in etiology and pathology of ketosis. We hypothesized that ketotic cows will display a different serum NEFA composition compared to healthy controls, and fatty acid related indicators for ketosis prediction can be screened. Pre-partum healthy cows were recruited, and blood samples were collected on −7, 3, 7, 14 and 21 d postpartum. Cows were further divided into a healthy control group (C group, n = 6) and a ketosis group (K group, n = 6) if blood β-hydroxybutyric acid levels exceeded 1.2 mm during the experiment. NEFA composition was then analyzed by means of Gas Chromatography-Mass Spectrometer (GC-MS). Only C12 : 0% was significantly higher in C group than K group on 7 d pre-partum (P < 0.05), when the cows were not diagnosed with ketosis. Five fatty acids displayed statistical differences in composition between C and K group (P < 0.05), namely C12 : 0, C16 : 0, C17 : 0, C18 : 1n9 and C22 : 1n9. Saturates%, unsaturates%, mono-unsaturates% and saturates/unsaturates were also different between C and K group (P < 0.05). Of note, C18 : 1n9/C12 : 0 and C18 : 1n9/C22 : 1n9 in K group were significantly higher than those in controls on 7 d pre-partum (P < 0.05). It is suggested that the ratios show potential as indicators for prediction of ketosis.