In the present work, a two-photon excited fluorescent chemosensor for Cu(2+) was prepared. The probe was constructed on the basis of internal charge transfer (ICT) principle with macrocyclic dioxotetraamine as the Cu(2+) receptor. The good water-solubility of the molecule enabled recognition and assay of Cu(2+) ions in biological media. The photophysical properties of the chemosensor were investigated in detail, exhibiting favorable fluorescence quantum yield and moderate two-photon absorption cross-section. The studies on binding thermodynamics demonstrated the formation of 1 : 1 complex between the chemosensor and Cu(2+) and an association constant of ca. 1.04 × 10(5) M(-1). Due to the rational design of the molecular structure, the sensor was highly specific to Cu(2+), which ensured high selectivity in Cu(2+) determination. Upon Cu(2+) binding, the intramolecular charge-transfer extent within the chromophore was weakened resulting in a remarkable quenching of fluorescence, based on which quantitative determination of Cu(2+) was performed. Good linearity was obtained between the fluorescence quenching value and Cu(2+) concentration ranging from 0.04 to 2.0 μM in aqueous solution. Benefiting from the merits of two-photon excitation, the chemosensor was free of interference from background luminescence in serum. A homogeneous quantitative determination of Cu(2+) was achieved in the serum medium with a linear range of 0.04 to 2.0 μM. Considering the structural flexibility of the sensor, this work also opens up the possibility to construct other two-photon excited chemosensors for direct homogeneous assay of various molecules/ions in complicated biological sample matrices.