A carbon dot (C-Dot)-based two-photon fluorescent probe has been developed for the monitoring of pH changes across a broad range with high sensitivity and selectivity. The inorganic-organic probe also shows good biocompatibility and cell permeability, and thus can be successfully applied in bioimaging and biosensing of physiological pH in living cells, as well as living tissues at a depth of 65-185 μm. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.