Abstract: The high-fluorescence functionalized graphene quantum dots (Cys-Pro-GQD) were prepared by one-step pyrolysis of a mixture containing L-cysteine, L-proline, and citric acid. Cys-Pro-GQD was then conjugated with the tobramycin (TOB) aptamer (Apt) and subsequently combined with graphene oxide (GO) to construct a fluorescent aptasensor (GO/Apt-Cys-Pro-GQD) system, in which the fluorescence was quenched. When TOB existed in the sample, TOB competed with GO to bind to Apt-Cys-Pro-GQD, and the fluorescence of the system was recovered. Based on this, the title research was carried out. 2.0 g of the milk sample was taken, and 5 mL of 5% (volume fraction) phosphoric acid solution, 1.0 mL of 300 g·L⁻¹ zinc sulfate solution, and 0.20 mL of trifluoroacetic acid were added. The mixture was made its volume up to 10 mL with water, then shaken, centrifuged, and filtered. 100 μL of the filtrate was added to the GO/Apt-Cys-Pro-GQD system. TOB in milk was determined by fluorescence spectrometry at the excitation wavelength of 350 nm and the emission wavelength of 440 nm. As shown by the results, linear relationship between the logarithms of TOB concentration and the corresponding fluorescence intensity was kept in the range of 50-5 000 nmol·L⁻¹, with detection limit (3.143s) of 10 nmol·L⁻¹. Test for recovery was made by the standard addition method, giving results in the range of 90.9%-108%, with RSDs (n=6) of the determined values in the range of 1.6%-2.5%.