Abstract: A rapid, sensitive and high throughput micro-plate method was established to quantify polysaccharides in this study. Anthrone-sulfuric acid micro-plate assay in a certain reaction was tested. A single test and a response surface methodology was applied to design and optimize four factors including reaction time, incubation temperature, anthrone concentration and the amount of anthrone in order to maximize R2 and minimize RSD in each standard curve which obtained in different conditions. It was demonstrated that the order of significant impacting R2 is incubation temperature, anthrone concentration, reaction time and the quantity of anthrone add to while the order of significant impacting RSD is incubation temperature, anthrone concentration, the amount of anthrone and reaction time. Preferred reactive condition is be discovered and the best incubation temperature is 96°C, the best reaction time is 36 min, anthrone concentration is 1.1 mg mL-1 and the amount of anthrone is 150 μL. The detectable range of this method is 20-260 μg L-1 under the optimized condition. Recovery test showed high accuracy and practicability in this method by recovery rate ranged in 99.5-100.6%. The method was successfully applied to quantify polysaccharides of different source and the method has high resistance to interference of protein during measurement process.