G-quadruplex (G4) is a noncanonical nucleic acid secondary structure that has implications for various physiological and pathological processes and is thus essential to exploring new approaches to G4 detection in live cells. However, the deficiency of molecular imaging tools makes it challenging to visualize the G4 in ex vivo tissue samples. In this study, we established a G4 probe design strategy and presented a red fluorescent benzothiazole derivative, ThT-NA, to detect and image G4 structures in living cells and tissue samples. By enhancing the electron-donating group of thioflavin T (ThT) and optimizing molecular structure, ThT-NA shows excellent photophysical properties, including red emission (610 nm), a large Stokes shift (>100 nm), high sensitivity selectivity toward G4s (1600-fold fluorescence turn-on ratio) and robust two-photon fluorescence emission. Therefore, these features enable ThT-NA to reveal the endogenous RNA G4 distribution in living cells and differentiate the cell cycle by monitoring the changes of RNA G4 folding. Significantly, to the best of our knowledge, ThT-NA is the first benzothiazole-derived G4 probe that has been developed for imaging G4s in ex vivo cancer tissue samples by two-photon microscopy techniques.