A method based on hollow fiber supported liquid membrane extraction coupled with a gas chromatograph equipped with flame ionization detector (GC-FID) was developed for the determination of six short-chain fatty acids including acetic acid, propionic acid, i-butyric acid, n-butyric acid, i-valeric acid and n-valeric acid in serum. Hollow fiber supported liquid membrane extraction was employed for preconcentration and clean-up of the samples. The fatty acids were extracted from the acidic donor (diluted serum) into a liquid membrane formed in the wall of the hollow fiber with 10% tri-n-octylphoshphine oxide (TOPO) in di-n-hexyl ether, and then extracted back into a basic acceptor solution filled in the lumen of the hollow fiber. After being acidified with HCl, the acceptor was directly analyzed by GC-FID. The acceptor concentration, donor pH, membrane liquid and extracting time were optimized giving an enrichment factor up to 155 times. The good linearity (r(2)>0.980), reasonable recovery (87.2-121%), and satisfactory intra-assay (8.2-11.5%) and inter-assay (6.1-11.6%) precision illustrated the good performance of the present method. Limits of detection (LOD) ranged from 0.04 to 0.24 microM and limits of quantification (LOQ) varied from 0.13 to 0.80 microM.