Molecular imprinting technology (MIT) and Mn-doped ZnS room-temperature phosphorescent quantum dots (Mn-ZnS RTP QDs) were combined to create a molecularly imprinted polymer (MIP) (MIP-coated QDs) that could specifically identify malachite green (MG). The MIP-coated QDs and the target MG small-molecules formed a phosphorescent resonance energy transfer system, and the RTP intensity was weakened with the increasing MG concentration. The MG detection limit and detection range were 30.34 nM and 0.08–80 μM, respectively. The spiked recovery rates in MG detection from fish meat and natural water were 96–107% and 95–102%, respectively. The MIP-coated QDs integrated the RTP and the high selectivity of MIT, and avoided interference from the background fluorescence or scattering light in biological or environmental samples and from competitive substances. Thus, the new MIP-coated QDs can be used to specifically detect trace MG in fish meat or natural waters.