A new two-photon fluorescent probe, ADNO, for nitric oxide (NO) based on intramolecular photoinduced electron transfer (PET) mechanism displays a rapid response to NO with a remarkable fluorescent enhancement in PBS buffer. The excellent chemoselectivity of ADNO for NO over other ROS/RNS (reactive oxygen species or nitrogen species) and common metal ions was observed. Moreover, ADNO has been successfully applied in fluorescence imaging of NO of living cells using both one-photon microscopy (OPM) and two-photon microscopy (TPM).A new two-photon fluorescent probe is capable of monitoring NO through a turn-on fluorescence signal output (Φ/Φ0 up to 8-fold) both in aqueous media and living cells.