Cholesterol is an important biological molecule with many important functions. However, high serum cholesterol is a health hazard. Thus analysis of cholesterol is important and many analytical techniques have been developed. The objectives of the proposed research are to develop an economical, rapid method for the determination of total cholesterol with good selectivity and enhanced sensitivity. For evaluation of proposed flow injection analysis with peroxyoxalate chemiluminescence (FIA-POCL) method, figures of merit such as accuracy, precision, and linear dynamic range will be assessed. The proposed procedure was then applied to the determination of total cholesterol in foods. The procedure was linear for cholesterol from 0.01 to 0.120 mg/mL. The relative standard deviation was 2.57%. The recoveries were 97.5-103.3% for commercial standard cholesterol sample, and 101.5-108.0% for butter. The proposed method was applied to analysis of cholesterol in food and the results were consistent with expected values.