In fluorescence imaging and detection, undesired fluorescence interference (such as autofluorescence) often hampers the contrast of the image and even prevents the identification of structures of interest. Here, we develop a quantum coherent modulation-enhanced (QCME) single-molecule imaging microscopy (SMIM) to substantially eliminate the strong fluorescence interference, based on manipulation of the excited-state population probability of a single molecule. By periodically modulating the phase difference between the ultrashort pulse pairs and performing a discrete Fourier transform of the arrival time of emitted photons, the decimation of single molecules from strong interference in QCME-SMIM has been clearly determined, where the signal-to-interference ratio is enhanced by more than 2 orders of magnitude. This technique, confirmed to be universal to organic dyes and linked with biomacromolecules, paves the way to high-contrast bioimaging under unfavorable conditions.