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Phenolic compounds, including anthocyanins have attracted much attention due to their biological activities. In this work, the liquid-liquid organic solvent extraction successfully prepared the phenolic and anthocyanin-rich fractions of muscadine grapes. The individual phenolics and anthocyanins were identified by high-performance liquid-chromatography mass spectrometry (HPLC-MS). Also, HPLC with evaporative light scattering detector (ELSD) for the simultaneous determination of different groups of phenolics in muscadine grapes was established. Besides the strong antioxidant activity of muscadines, anti-diabetic and anti-cancer activities of muscadine extracts were investigated as well. Results showed that the phenolic and anthocyanin-rich extracts obeyed the competitive inhibition mode against three selected key enzymes, which suggested that the muscadine extracts possessed the strong anti-diabetes activity. And the cell proliferation assay indicated that the phenolic and anthocyanin-rich fractions of muscadines exhibited the strong anti-proliferative activity on cancel cells rather than other fractions. Therefore, health-promoting properties of muscadine grapes were confirmed.