A two-photon active probe for physiological copper (Cu2+) detection is expected to play an important role in monitoring biological metabolism. Herein, a novel Schiff base derivative (E)-2,2'-((4-((4-(diethylamino)-2-hydroxybenzylidene)amino)phenyl)azanediyl)bis(ethan-1-ol) (L) with remarkable two-photon activity was developed and synthetically investigated. L presents high selectivity and sensitivity for Cu2+ sensing in ethanol/HEPES buffer (v/v, 1 : 1), which is accompanied by the fluorescence switching "off" and subsequently "on" with the addition of EDTA. The mechanism for the detection of Cu2+ is further analyzed using 1H NMR titration, mass spectra and theoretical calculations. Furthermore, since the probe L possesses good photophysical properties, excellent biocompatibility and low cytotoxicity, it is successfully applied to track Cu2+ in the cellular endoplasmic reticulum by two-photon fluorescence imaging, showing its potential value for practical applications in biological systems.