A new two-photon fluorescent probe (MCN) for viscosity imaging was developed based on a 6-substituted quinoline framework. MCN showed an excellent "off-on" fluorescence response (ca. 90-fold enhancement) with viscosity increasing in the glycerol-water viscosity system. MCN showed great sensitivity to viscosity (R2 = 0.98, x = 0.65), which gave rise to cell imaging for micro-viscosity or real-time cell imaging during apoptosis with low cytotoxicity under two-photon excitation (λex = 800 nm). Fluorescence lifetime imaging (FLIM) of living HeLa cells stained with MCN revealed that the intracellular average viscosity value was 73.45 ± 21.55 cP in cytosol. Imaging in living tissue slices indicated that MCN can work in deep tissue (∼130 μM) under two-photon excitation. Moreover, MCN also showed the capacity for in vivo imaging viscosity in zebrafish with obvious fluorescence emission.