Chemiluminescence (CL) emission from luminol-tetrachloroaurate ([AuCl(4)](-)) system studied in presence of monosaccharide sugars such as glucose and fructose was investigated on a microfluidic chip fabricated by the soft lithography technique. CL emission from the luminol-[AuCl(4)](-) system at 430 nm was intensified remarkably by the catalytic activity of glucose and fructose at room temperature. Under optimized conditions, the CL emission intensity of the system was found to be linearly related to the concentration of the sugars. Based on this observation, nonenzymatic determination of total sugar (glucose, fructose, or hydrolyzable sucrose) was performed in a rapid and sensitive analytical method. The results revealed that the linearity ranged from 9 to 1,750 μM for glucose and 80 to 1,750 μM for fructose, with a limit of detection of 0.65 and 0.69 μM, respectively. The relative standard deviations determined at 250 μM based on six repetitive injections were 1.13 and 1.15% for glucose and fructose, respectively. The developed method was successfully applied for determination of the total sugar concentration in food and beverages.