Conjugated linoleic acid (CLA) is a term representing a mixture of positional and geometric isomers of octadecadienoic acid with a conjugated double bond system. Conjugated linoleic acid has attracted a great deal of interest among nutritionists because it is a natural fat component that appears to have a number of health improvement properties. The cis-9, trans-11-CLA is the major CLA isomer found in dairy products accounting for 75% to 90% of the total CLA in milk fat. A capillary gas chromatographic method equipped with a flame ionization detector for the analysis of the cis-9, trans-11-CLA in milk fat was developed. The cis-9, trans-11-CLA was extracted with hexane-isopropanol, methylated with methanol-sodium methylate and cis-9, trans-11-CLA was separated and quantified using gas chromatography. Retention time of the peaks was used for qualitative analysis, while external standard method was used for quantitative analysis. The recovery of the cis-9, trans-11-CLA was 100.26%. The relative standard deviation was 1.9% (n = 6). This method presented is advantageous for high precision, high sensitivity analysis with smaller sample size and simpler pretreatment. It would be of significance for analyzing the contents of other fatty acids in the milk and milk products.