We developed an automatic QuanTi-FRET (AutoQT-FRET) method to measure the G and β factors by performing single imaging. The method for transfecting multiple plasmids in one cell dish reduces the time and shortens the process of calculating the correction factor G of the system. Simultaneously, the AutoQT-FRET method can classify cells transfected with multiple standard FRET plasmids in a cell petri dish. Thus, it enables the accurate measurement of the system correction factors G and β values using the one-button operation of one-time imaging of a cell petri dish, which greatly simplifies data processing and lowers the threshold for researchers who use quantitative FRET measurements. The AutoQT-FRET method is not only robust but also fast and concise.