Dopamine, a neurotransmitter with multiple functions in the brain. The abnormal of dopamine levels are generally considered to be associated with various nervous system diseases. Herein, a novel ‘‘off–on” single-excited dual-emissive ratio luminescent aptasensor for highly sensitive and accurate monitoring of DA levels has been prepared via electrostatic interaction connecting persistent luminescent nanoparticles (PLNPs) ZnGaO:Sm and ZnGeO:Mn with different emission wavelengths. ZnGaO:Sm@Cy5.5-ZnGeO:Mn (ZGS@Cy5.5-ZGM), this ratiometric aptasensor exhibits two-emission peaks at 533 and 700 nm under a single-excitation wavelength of 254 nm. The afterglow luminescence of ZnGaO:Sm-apt@Cy5.5 at 700 nm is effective quenched by complementary chains of Cy5.5-modified DA aptamers with 710 nm specific absorption and only efficiently recovered by DA; meanwhile, the afterglow luminescence of ZnGeO:Mn as reference at 533 nm remains unchanged. The as-designed autofluorescence-free and exogenous-free interference ratiometric sensing system ZGS@Cy5.5-ZGM showed high selectivity for DA in complex biological samples. Under optimal detection conditions, the luminescence intensity ratio Δ(I700/I533) showed a good linear relationship with the concentration of DA in the range of 0.02–80 μM and the detection limit as low as 4 nM. The recoveries of DA in serum, urine and sweat were 97.4 % to 105.3 %, 96.4 % to103.4 %, and 95.1 % to 104.4 %, respectively. Current strategies based on PLNPs ratiometric sensors provide an effective method with high selectivity and accuracy for the detection of DA in clinical samples.