The tea-like beverage Stevia rebaudiana Bertoni (Stevia) is popular in China because it reduces blood glucose and has a sweet taste. In this work, a comprehensive quality assessment of Stevia led to the discovery of five phenylethanoid glycosides, namely steviophethanoside (1), cuchiloside (2), salidroside (3), icariside D (4), and tyrosol (5). Of them, compound 1 is a novel compound. Mass spectrometry and NMR spectroscopy were employed to confirm the absolute configuration. A hydrolytic step with 4 N TFA at 95 °C for 4 h was used to confirm the monosaccharides. In addition, Discovery Studio 4.0 was used to predict the ADME and toxicity activity of compound 1. The results suggested that compound 1 was biocompatible and had poor toxicity, which was verified by rat INS-1 islet β cells through an MTT assay. Meanwhile, a significant stimulatory effect on INS-1 cells was observed, which indicated a hypoglycemic effect of compound 1. This is the first report that describes a natural, novel, and hypoglycemic phenylethanoid glycoside in Stevia.