Steviol glycosides, mostly extracted from Stevia rebaudiana Bertoni, are non-caloric, safe natural sweeteners. Steviosides make up more than 50% of total steviol glycosides but possess a bitter aftertaste, which limits their application as food additives. Trace amounts of rubusosides are acceptable sweeteners, but their supply is limited. In this study, we screened an endophytic bacteria (Lelliottia LST-1) that can specifically convert stevioside into rubusoside and reach a conversion rate of 75.4% after response surface optimization. Potential β-glucosidases GH3-1, GH3-2, GH3-3, and GH3-4 based on whole genome sequencing were expressed and interacted with steviol glycosides to explore principle and process of biological transformation. High-performance liquid chromatography revealed that all enzymes hydrolyzed stevioside and generated rubusoside, but substrate specificity analysis indicated that GH3-2 is the most suitable choice for the durative, steady, and high-efficiency production of rubusoside