Goji (Lycium barbarum L.) leaves are widely processed as a healthy tea (GT) in China for their multiple biological activities. In this study, different polar segments from GT ethanolic extract were obtained using the liquid-liquid fractionation method. n-Butanol fraction (GN) had the highest antioxidant activity while ethyl acetate fraction (GE) showed the highest inhibitory effect on the α-glucosidase enzyme. The anti-diabetic effect of GE was further investigated in type 2 diabetic mellitus (T2DM) mice. The oral administration of GE (300 or 600 mg/kg) reduced fasting blood glucose levels, improved glucose tolerance ability, and ameliorated insulin resistance in T2DM mice. GE intervention could enhance the cholesterol transportation in the serum in coincidence with decreased total cholesterol, triglyceride levels, and non-esterified fatty acid. Transcriptomic, immunofluorescence and Western blotting analysis revealed that GE significantly up-regulated insulin receptor substrate 1, phosphatidylinositol-3-kinase, protein kinase B, glycogen synthesis, and glucose transporter 4. AMP-activated protein kinases mediate gluconeogenesis and glycogenolysis, such as phosphoenolpyruvate carboxy kinase 1 and glucose-6-phosphatase in the liver and kidney tissues of GE groups were decreased. Thus, GE alleviated the liver, kidney, and pancreatic tissue damage. The phytoconstituents of GE were characterized, and rutin, neochlorogenic acid, and its acylated derivatives have hypoglycemic effects. Furthermore, these findings suggested that GT might be used as a promising hypoglycemic candidate.