Abstract: The formula of Rosa roxburghii Tratt sparkling water was optimized by orthogonal tests, whereas the scavenging rates of hydroxyl free radical and superoxide anion free radical were determined. The anti-fatigue activity was observed by weight-bearing swimming test in mice. The results showed the optimized formula as below: fructose syrup 8%, R. roxburghii juice 7%, carbon dioxide 0.4%, taurine 0.05%, guarana extract 0.05%,citric acid 0.006%, sodiumphytate 0.005%, sucralose 0.000 9%. The product prepared according to the optimized formula was light yellow and transparent, which had uniform color, moderate taste of acidity and sweetness with the special aroma of R. roxburghii as well as the cool feeling of sparkling beverage. The content of vitamin C was290 mg/100 mL, and the scavenging rates of hydroxyl radical and superoxide anion radical were 92.62% and82.84%, respectively. The weight-bearing swimming time of the product reached 26.25 s, showing strong anti-fatigue activity. The combination of R. roxburghii juice, taurine and guarana extract can significantly improve the anti-fatigue effect of the product.