Though copper ion (Cu²⁺) is a widely distributed pollutant in the water environment, it plays a vital role in many biological processes. Hence, rapid detection and identification of Cu²⁺ are important. In the past few years, fluorescence sensing has become the golden standard to detect Cu²⁺, due to its high sensitivity, high selectivity, and useful applications in biology, medicine, environment and chemistry. Thus, researchers have widely concerned with the design and synthesis of Cu²⁺ fluorescent probes. In this study, a novel probe 2a with high sensitivity and selectivity for detecting Cu²⁺ is designed. It is illustrated that 2a is a ratiometric fluorescent probe, which recognizes Cu²⁺ by a Förster resonance energy transfer (FRET) mechanism. Meanwhile, the two-photon absorption (TPA) optical properties of 2a are calculated. The calculated results demonstrate that 2a possesses a higher energy transfer efficiency upon excitation and a larger TPA peak in the near-infrared region than others. Therefore, it can be inferred that the probe 2a should be an excellent two-photon (TP) excited FRET-based ratiometric fluorescent probe for Cu²⁺. The detailed investigations can provide a theoretical basis to synthesize copper-ion-responsive TP FRET-based ratiometric fluorescent reagents, which are powerful tools for the two-photon microscopy (TPM) and biological imaging of Cu²⁺ in vivo.