G4s play a very important role not only in human biological systems but are associated with tumor formation, which is considered as a novel target for the exploitation of anti-tumor drugs. Therefore, it is essential to develop near-infrared (NIR) fluorescent probes to study G4s. In this article, we designed a large stokes shift probe (AOC) which can be activated NIR fluorescent by G4s in living cells and tumor tissue. In vitro experiments shown that AOC exhibited no fluorescence emission in aqueous solution, while significantly enhanced at 768 nm in the presence of the G4s. The optimal excitation wavelength of the probe is about 640 nm, indicating that the probe AOC has a large stokes shift (128 nm). Meanwhile, AOC is dynamically responsive to the concentrations of DNA G4s (22AG, Mito-160) and exhibits high selectivity toward DNA G4s with desirable stability in the physiological pH conditions. Imaging studies of different cell lines have shown that cancer cell lines fluoresced brightly than normal cell lines, implication that the expression of G4s may be higher in tumor cell lines. We further performed fluorescence imaging of tumor tissues using AOC, which showed that tumor tissues had the brightest fluorescence intensity relative to normal organ tissues. These experimental results further suggest that the NIR fluorescent probe may have tumor diagnostic properties with great potential for application.