As a kernel attribute of molecular semiconductor, the energy gap fundamentally determines the working principle which is crucial to the device performance optimization, such as the photoelectric conversion efficiency of organic solar cells. Therefore, a precise quantification for energy gap of a molecular semiconductor is crucial. A three-terminal vertical structure (Al/AlOX/Au/ molecular semiconductor/Al), named hot electron transistor has been verified to be the most powerful method for energy gap determination. The Al/AlOX/Au tunneling junction is used for energy regulation and the Au/ molecular semiconductor/Al diode is applied for providing an authentic device. By monitoring the charge transport behaviors in the real device, the electron injected barrier (or LUMO) and hole injected barrier (or HOMO) could be obtained, which makes for the interfacial energy gap determination. This research develops a handy tool for the ordinary research groups working in molecular electronics. It will contribute to the rapid development of molecular electronics.