G‐quadruplex DNA show structural polymorphism, leading to challenges in the use of selective recognition probes for the accurate detection of G‐quadruplexes in vivo. Herein, we present a tripodal cationic fluorescent probe, NBTE, which showed distinguishable fluorescence lifetime responses between G‐quadruplexes and other DNA topologies, and fluorescence quantum yield (Φf) enhancement upon G‐quadruplex binding. We determined two NBTE‐G‐quadruplex complex structures with high Φf values by NMR spectroscopy. The structures indicated NBTE interacted with G‐quadruplexes using three arms through π–π stacking, differing from that with duplex DNA using two arms, which rationalized the higher Φf values and lifetime response of NBTE upon G‐quadruplex binding. Based on photon counts of FLIM, we detected the percentage of G‐quadruplex DNA in live cells with NBTE and found G‐quadruplex DNA content in cancer cells is 4‐fold that in normal cells, suggesting the potential applications of this probe in cancer cell detection. G4 imaging: A tripodal cationic fluorescence probe, NBTE, was used for the visualization (based on fluorescence lifetime) and quantification (based on photon counts) of G‐quadruplex DNA in live cells by fluorescence lifetime imaging microscopy (FLIM). Structural studies provided a structural basis for the DNA‐topology‐based fluorescence response of NBTE.