The samples collected from 11 areas of Guizhou Province, China, were investigated to illustrate the effects of soil indicators and environmental factors on flavonoids in R. sterilis. A facile method for the simultaneous determination of the main flavonoids in R. sterilis fruits was established with high performance liquid chromatography. The major flavonoids, accounting for 1.13-5.98% of the total flavonoids, consist of rutin (1.05-5.58%), quercetin (0.02-0.09%), and kaempferol (0.06-0.31%). Moreover, four local environmental factors and eleven soil indicators were determined from each planting area. The results of variance analysis showed significant differences in soil properties from different planting areas. Multivariate analysis (redundancy analysis and partial correlation analysis) indicated that sunshine intensity and annual precipitation were the major environmental factors for the content of flavonoids in the fruits, while soil organic matter (SOM), pH, available nitrogen (AN), total phosphorus (TP) and available potassium (AK) are basic and essential nutrients for plants growth that influence the content of flavonoids in R. sterilis. The results revealed that sunlight intensity was the main factor for the content of flavonoids in the fruits by affecting the synthesis rate. The soil indicators, such as SOM, pH, AN, TP and AK, should be adequate and balanced, which supply essential nutrients to contribute to maintaining health and the content of active substances in R. sterilis.