The interfacial delamination or debonding induced by local stress concentration is one of the kernel factors leading to spalling failure of thermal barrier coatings (TBC). In this work, for the rapid and accurate characterization of the interface delamination and debonding in TBC, the detection method and instrument were investigated based on fluorescence imaging. First, based on the principle of reflection-enhanced fluorescence, an experimental method was presented to detect interfacial debondings in TBC. A coaxial fluorescence-imaging instrument was established to realize the method above. In addition, an alkaline soluble method (ASM) was presented in this work to prepare prefabricated debondings with different transverse sizes, shapes, and thicknesses, which are basically consistent with the interfacial debondings in an actual TBC structure. Through a series of experiments, this paper verified that, using the proposed method and optical instrument, the resolutions to detect internal debonding are as small as 6 µm in the thickness dimension and less than 0.3 mm in the transverse dimension.