A novel ratiometric fluorescence molecularly imprinted sensor based on a dual-emission quantum dot hybrid was fabricated and used as an alternative analytical tool for the detection of tetracycline. In the synthesis process, red-emitting quantum dots (r-QDs) and green-emitting quantum dots (g-QDs) were modified by two different methods. Afterward, a stepwise precipitation polymerization imprinting reaction was performed to prepare the novel ratiometric fluorescence molecularly imprinted sensor (MIP-g/r-QD sensor). The MIP-g/r-QD sensor integrated the advantages of molecularly imprinted polymers and ratiometric fluorescence probes. The specific recognition sites in the polymer layers could adsorb tetracycline molecules, and then they caused fluorescence quenching behavior of g-QDs via an electron transfer process. Under the optimal conditions, a linear relationship was obtained covering the range from 10 to 160 μmol/L, with a correlation coefficient of 0.9976 and a high imprinting factor of about 3.3. Moreover, the novel MIP-g/r-QD sensor was successfully applied to detect tetracycline in milk samples. This work provides a new way to fabricate an efficient ratiometric fluorescence molecularly imprinted sensor based on quantum dots for convenient, fast, and highly selective and sensitive detection of organic molecules. Graphical abstract A novel ratiometric fluorescence molecularly imprinted sensor based on a dual-emission quantum dot hybrid was fabricated and used as an alternative analytical tool for the detection of tetracycline. AIBN azoisobutyronitrile, EGDMA ethylene glycol dimethacrylate, KH-570 3-(methacryloyloxy)propyltrimethoxysilane, OVDAC octadecyl-4-vinylbenzyldimethylammonium chloride, PDDA poly(diallyldimethylammonium chloride), QD quantum dot, TEOS tetraethoxysilane.