In this study, we have developed a fluorescence chromatographic assay for the quantification of total cholesterol in serum, which is a well-known risk predictor for cardiovascular diseases. The new assay system consists of a chromatographic strip in a cartridge, enzyme buffer containing cholesterol esterase, cholesterol oxidase, horseradish peroxidase, and color developer AEC, and a laser fluorescence scanner. The correlation coefficient (r) between cholesterol concentration and relative fluorescence units was 0.968 in the new assay, showing a reliable linearity through the tested range of cholesterol. Recovery test and comparability with a Hitachi 747 instrument showed 106.5-94% and r = 0.939 (p<0.001), respectively. The new assay system for cholesterol was developed as a pre-POCT platform conducted in clinics since it is fast (8 min) and uses a small volume of sample (5 μl), and it may be applied for on-site diagnostics to replace expensive automated biochemical analyzer.