Current methods to monitor cellular ATP do not provide spatial or temporal localization of ATP in single cells in real time or they display imperfect specificity to ATP. Here, we have developed a single cell, Enhanced Acceptor Fluorescence (EAF)-based ATP biosensor to visualize ATP in real time. This biosensor utilizes a modified mimic of the epsilon-subunits of the Bacillus subtilis F0F1 synthase and is coupled to the EAF fluorophores pairs, GFP and YFP. The sensor was then used to monitor ATP in a heterogeneous glioblastoma multiform cancer cell population. We anticipate that this innovative technology and our better understanding of the ATP machinery will have substantial influence on future translational studies.