The sodium ion plays critical roles in many physiological and pathological processes such as nerve conduction and muscle and heart contraction; it modulates the electrolyte level, cation transport, and cell volume. Therefore, it is very important to develop two-photon fluorescence probes for three-dimensional dynamic imaging of sodium ions in cells. A novel two-photon fluorescence probe (DNa) for Na derived from dicyanostilbene as a two-photon fluorophore and benzo-15-crown-5 as a novel Na ligand was develped, and its structure was analyzed and identified. DNa possessed small molecule size, large two-photon absorption cross-section (1054 GM), noncytotoxic effect, excellent photostability, moderate water-solubility, and good cell-permeability. DNa showed excellent selectivity for Na and was not interfered by other ions. The dissociation constant of Na , kd = 23 ? 2 mmol L-1. DNa can be used in the three-dimensional imaging of Na in cells, so it is an excellent two-photon fluorescence probe.