This study described a simple and quick method to detect trace quantities of a non-reducing sugar (viz. sucrose) in the root exudates of cucumber (Cucumis sativus) under CO2 enrichment. Sucrose was determined by analyzing fructose and glucose before and after invertase digestion using high-performance liquid chromatography. Using this technique, the optimal hydrolysis condition was 5.00 μg·mL−1 invertase for 10 minutes. The detection limit of ultraviolet-visible detector by post-column derivatization with tetrazolium was 0.25, 0.43, 0.48, and 1.95 μg·mL−1 for fructose, glucose, sucrose, and maltose, respectively, and sensitive enough for determination of sugars in root exudates. The dry weight of cucumber at the seedling stage (19 days after transplant) increased by 58.4% when the CO2 level was elevated from 380 to 1200 μmol·mol−1, whereas the differences were not significant at the initial fruiting stage (63 days after transplant). The photosynthesis rate in 1200 μmol·mol−1 CO2 was 58.0% higher than that in 380 μmol·mol−1 CO2 at the seedling stage and 74.2% higher at the initial fruiting stage. Total amount of sugars in cucumber root exudates was significantly increased with increasing CO2 concentration. The total sugars in root exudates increased by 130.4% and 102.3% in 1200 μmol·mol−1 CO2 compared with that in 380 μmol·mol−1 CO2 at seedling and initial fruiting stages, respectively. Elevated CO2 altered sugar composition in root exudates. Sugars in root exudates released per plant were significantly higher at the initial fruiting stage than that at the seedling stage, whereas the differences in sugars released per gram of root tissue between these two growth stages were not significant. Our results suggest that sugars were increased only in as much as root mass increased. This study provides a simple and quick method to detect 1 to 500 μg·mL−1 sugars in root exudates, and the results illustrate the variation in the sugar composition in cucumber root exudates among the CO2 levels and growth stages.