In contrast to one-photon microscopy, two-photon probe-based fluorescent imaging can provide improved three-dimensional spatial localization and increased imaging depth. Consequently, it has become one of the most attractive techniques for studying biological events in living cells and tissues. However, the quantitation of these probes is primarily based on single-emission intensity change, which tends to be affected by a variety of environmental factors. Ratiometric probes, on the other hand, can eliminate these interferences by the built-in correction of the dual emission bands, resulting in a more favorable system for imaging living cells and tissues. Herein, for the first time, we adopted a through-bond energy transfer (TBET) strategy to design and synthesize a small molecular ratiometric two-photon fluorescent probe for imaging living cells and tissues in real time. Specifically, a two-photon fluorophore (D-π-A-structured naphthalene derivative) and a rhodamine B fluorophore are directly connected by electronically conjugated bond to form a TBET probe, or Np-Rh, which shows a target-modulated ratiometric two-photon fluorescence response with highly efficient energy transfer (93.7%) and two well-resolved emission peaks separated by 100 nm. This novel probe was then applied for two-photon imaging of living cells and tissues and showed high ratiometric imaging resolution and deep-tissue imaging depth of 180 μm, thus demonstrating its practical application in biological systems.