The related substances in Entecavir Oral Preparation were separated on a Thermo C18 column（150 mm×2.1 mm×2 μm） by gradient elution，with methanol-0.2%formic acid as mobile phase at a flow rate of 0.3 mL/min and a column temperature of 30 ℃，and the detection wavelength was set at 254 nm.The proposed method was used to detect the related substances in Entecavir Oral Preparations from different pharmaceutical manufacturers.The chemical structures of related substances were analyzed by LC-MS/MS.Two structures of the related primary substances were identified，which both belonged to the isomers of entecavir.This study provided an important basis for the quality and stability control of this product.