The present disclosure relates to a two-photon probe for in-vivo imaging, having high selectivity for copper(II) ion, and a method for quantitatively estimating copper(II) on (Cu2+) in vivo using the same. The two-photon probe according to the present disclosure can detect intracellular free copper(II) on for a long period of time with a penetration depth greater than 90 μm in living cells and tissues without the problems of mistargeting and photobleaching and can quantitatively estimate copper(II) on concentration in vivo using the ratio of blue emission intensity at 400-450 nm and red emission intensity at 550-650 nm.