A two-photon probe for real-time monitoring of intracellular magnesium ions is provided. Specifically, the two-photon probe is represented by Formula 1: wherein R is H or CH2OCOCH3. The two-photon probe is very suitable for real-time imaging of intracellular magnesium ions. The two-photon probe shows 17-fold two-photon excited fluorescence enhancement in response to Mg2+, which is 7-fold stronger than commercial probes, thus enabling staining of cells in a greatly reduced amount. In addition, the two-photon probe has a sufficiently low molecular weight to stain cells and is very suitable for monitoring Mg2+ ions present in the deep tissue. Furthermore, the two-photon probe can be effectively used for the quantitative as well as qualitative detection of intracellular magnesium ions. Further provided are a method for preparing the two-photon probe and a method for real-time monitoring of intracellular magnesium ions using the two-photon probe.