Disclosed are a glass substrate separation device and a glass substrate separation method. A plurality of first vacuum adsorption devices is adsorbed on one end of the glass substrate to separate the one end of the glass substrate and an OLED with an opening with a certain distance, and a metal wire is driven to enter the opening, and abuts a lower surface of the glass substrate to move to the other end of the glass substrate to accomplish the separation between the glass substrate and the OLED; and then, a second vacuum adsorption device is adsorbed at an intermediate position of the glass substrate to remove the glass substrate from the OLED to realize a glass substrate removal process after a LLO in an OLED module production, thereby avoiding a damage to the OLED and a PI layer disposed on a surface layer of the OLED.