Surface-initiated anionic-ring-opening multibranching polymerization was employed to prepare a hyperbranched polyglycerol (HPG) functionalized silica stationary phase for hydrophilic interaction liquid chromatography (HILIC). The obtained stationary phase was characterized by Fourier-transform infrared spectrometry (FT-IR) and thermogravimetric analysis (TGA). The chromatographic properties of the prepared stationary phase were systematically investigated. The abundance and multitude distribution of hydroxyl groups in HPG endowed the stationary phase with improved hydrophilicity and enhanced separation performance compared with the stationary phase functionalized with monolayer of hydroxyl groups. The stationary phase showed excellent retention of various polar compounds, such as nucleosides, necleobases, phenols and sulfanilamides, indicating great potential in the separation of complex biosamples.