To determine sugars content of agricultural products and foods, simultaneous quantitative analysis was carried out on fructose, glucose, sucrose, maltose, and lactose by HPLC-RI. Analysis conditions were set as column ZORBAX carbohydrate (4.6 mm ID×250 mm, 5 μm), the mobile phase of 75% ACN, the column temperature of 35oC, sample injection amount of 10 μL and the flow rate of 1 mL/min. Five standard solutions were isolated without interfering peak within 30 minutes and the calibration curves of standard were confirmed excellent linearity from 0.10% to 1.00% with R2≥0.999. Based on the chromatogram of the standard solution, the limit of quantification (LOQ) and the limit of detection (LOD) values were calculated. The accuracy of the analytical values were highest at 100% water extraction method to the fructose 95.7%, sucrose 98.7%, lactose 102.7% respectively, compared with reference value of a certified reference material (BCR644), by applying the four solvent extraction methods. Using an in-house quality control material (infant formula), repeatability and reproducibility values of this experiment were verified on the basis of AOAC guideline and reference values were set up at 1.17 g/100g of glucose, 0.85 g/100g of maltose, and 45.54 g/100g of lactose. Quality control charts were drawn up and used for sugars analysis of agricultural products.