We report on a novel dibenzothiophene-based two-photon fluorescent probe for selective nuclear bioimaging, which contains bilaterally symmetrical pyridine rings connected by a central conjugated-bridge dibenzothiophene. This probe possesses a large two-photon absorption cross-section of 471 GM, yields a 25-fold enhancement of the fluorescence titration, and a stronger photostability for nuclei labeling than existing probes. The real-time observation period is a minimum of 1800 s under a femtosecond laser excitation, which is significantly longer than that of 4′,6-diamidino-2-phenylindole. The above results confirm that this novel molecule is a suitable two-photon fluorescent probe for application to nuclear bioimaging in cells.