Dopamine and polyethyleneimine (PEI) copolymerized nanodots (PDA-PEI nanodots) with both fluorescence emission and quenching features were synthesized by a simple one-step reaction at room temperature. By adjusting the dopamine and PEI ratio as well as the chain length of PEI, the fluorescence emission and quenching properties of PDA-PEI nanodots can be controlled well. Under optimal conditions, the nanodots showed strong green fluorescence emission with an absolute quantum yield of 1-2% and a quenching efficiency of more than 99% to several fluorophores with emission wavelengths ranging from blue to red light regions. The nanodots with a large number of functional groups also showed strong affinity to nucleic acid strands, excellent solubility in aqueous solution, long-term stability, and uniform size distribution. Integrating these attractive features with the specific enzymatic digestion reaction of the DSN enzyme, a highly sensitive ratiometric fluorescence nanoprobe for miRNA analysis was developed. Aminomethylcoumarin acetate (AMCA), which possesses the same excitation wavelength but a well-resolved blue fluorescence emission with PDA-PEI nanodots, was selected as the signal-reporting unit for capture probe labeling, while the inherent green fluorescence of PDA-PEI nanodots served as the reference. According to the ratiometric fluorescence signal, the ratiometric fluorescence nanoprobes showed high sensitivity and good accuracy for the miRNA assay. Because of the high and universal quenching efficiency, stable fluorescence emission, easily assembled interface, and uniform morphology, the nanodots may have great application prospects to serve as a universal nanoplatform for the fabrication of ratiometric fluorescence nanoprobes.