A new carbazole tricationic salt,4,4’-(1E,1’E)-2,2’-(9-(2-(1-(2-hydroxyethyl)pyridinium-4-yl)ethyl)-9H-carbazole-3,6-diyl) bis(ethane-2,1-diyl) bis(1-(2-hydroxyethyl)pyridinium) iodide (THEPC) was synthesized. Photophysical experiments have shown that THEPC has large two-photon excited fluorescence action cross-sections (33 GM in the presence of DNA),which ranks THEPC as a good biological fluorophore. The results from electronic absorption,circle dichroism and single-/two-photon fluorescence emission spectra suggest that THEPC can strongly bind to DNA,with an intrinsic binding constant of 5.79 × 106 L mol-1. THEPC has better photostability under one-or two-photon excitation conditions. Finally,the staining photos from two-photon fluorescence microscopy (TPM) show that THEPC can exclusively label the nucleus with high contrast and without image distortion. These remarkable properties and optimized imaging ability make THEPC an attractive DNA probe in TPM. A new carbazole tricationic salt, 4,4 ’- (1E, 1’E) -2,2’ - (9- (2- (2- hydroxyethyl) pyridinium-4-yl) ethyl) -9H-carbazole Photophical experiments have shown that THEPC has a large two-photon excited fluorescence action cross (USP) -sections (33 GM in the presence of DNA), which ranks THEPC as a good biological fluorophore. The results from electronic absorption, circle dichroism and single- / two-photon fluorescence emission spectra suggest that THEPC can strongly bind to DNA, with an intrinsic binding constant of 5.79 × 106 L mol-1. THEPC has better photostability under one-or two-photon excitation conditions. Finally, the staining photos from two-photon fluorescence microscopy (TPM) show that THEPC can exclusively label the nucleus with high contrast and without image distortion. These remarkable properties and optimized imaging ability make THEPC an attractive DNA probe in TPM.