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Two‐photon excited fluorescence is a process in which the excited fluorophore relaxes and emits fluorescence after two‐photon absorption. Combined with appropriate two‐photon fluorescent probes, two‐photon microscopy enables observation deep inside tissue without damage. Former reports showed that the two‐photon absorption cross sections of donor–acceptor (D–A) dipoles, as well as D‐π‐D and D–A–D quadrupoles can be strengthened by using strong D–A groups. In this study, a novel water‐soluble fluorescent probe with D‐π‐A structure containing pyridine‐2,6‐dicarboxylic acid unit was synthesized. The maximum excitation wavelength and emission wavelength of the probe in aqueous solution were 328 nm and 471 nm, respectively. The fluorescence emitted by the probe can be quenched by various metal cations. The pyridyl N and carboxyl O in the probe formed chelates with Cu2+, which resulted in a fluorescence quenching response, and showed a linear relationship between fluorescent intensity and Cu2+ concentration with wide linear range and low limit of detection. CCK‐8 tests showed the probe had no obvious toxicity to 4T1 cells in the range of 0.4–50 μmol L−1, and the cell survival rate was above 86 %. The probe had a large two‐photon absorption cross section ranging from 680 nm to 820 nm. The two‐photon absorption cross section reached 100 GM at 760 nm of excitation wavelength. By means of microscopic imaging of 4T1 cells stained with the probe, one‐photon fluorescence represented blue emission, while two‐photon fluorescence represented bright green emission. The probe was proved to have good cell permeability, and preferred to target nucleus, showing application potential in two‐photon fluorescence imaging technology. [ABSTRACT FROM AUTHOR]