Quantum efficiency is a critical piece of information of a quantum emitter and regulates the emitter’s fluorescence decay dynamics in an optical environment through the Purcell effect. Here, we present a simple way to experimentally probe fluorescence quantum efficiency of single dibenzoterrylene molecules embedded in a thin anthracene microcrystal obtained through a co-sublimation process. In particular, we correlate the fluorescence lifetime change of single dibenzoterrylene molecules with the variation of the matrix thickness due to natural sublimation. With the identification of the molecule emission dipole orientation, we could deduce the near-unity intrinsic quantum efficiency of dibenzoterrylene molecules in the anthracene matrix.