A maleimide coupling naphthalimide was reported as new two-photon fluorescent (TPF) probe for cysteine (Cys). The probe was weakly fluorescent itself due to the donor-excited photoinduced electron transfer (d-PET). After reaction with Cys, d-PET process was blocked and fluorescence enhancement of the probe was observed at 470 nm. The d-PET principle was rationalized by theoretical calculations with density functional theory and time-dependent density functional theory. Thiol-maleimide addition between the probe and Cys was confirmed by H-1 NMR and mass spectrum measurements. TPF analysis demonstrated a 24.7-fold emission increase of the probe induced by Cys upon excitation at 760 nm. The two-photon action cross-section of probe-Cys adduct at 760 nm reached 42 GM compared to 1.7 GM for free probe. The probe showed high sensitivity and selectivity to Cys over other potential interferences; especially it had the capability to discriminate Cys from glutathione and homocysteine. Through TPF imaging, the probe was successfully applied in the detection of Cys in living cells. (C) 2014 Elsevier B.V. All rights reserved.