Objective: To achieve rapid, quantitative and high-throughput detection of aminoglycoside antibiotic residues in pork. Methods: Square-array aligned SERS substrates based on Au nanoflowers （AuNFs） were prepared using PP synthetic paper as a substrate. Pork samples were pre-treated by QuEChERs method and subjected to SRES. Results: Using 4-mercaptobenzoic acid as the SERS probe molecule, the substrate exhibited good homogeneity, SERS enhancement effect, reproducibility and stability. the SERS signal intensities at the characteristic peaks at 475, 619 cm^-1 showed good linear relationships with the logarithms of the concentrations of gentamicin sulphate and neomycin sulphate, respectively （R² of 0.991 6 and 0.990 7, respectively）. The limits of detections （LODs） were as low as 1×10^-9, 1×10^-8 mol/L, respectively, and were successfully applied to the rapid, quantitative and high-throughput determination of aminoglycoside antibiotics in pork. Conclusion: The experimental method provides an economical, efficient, time-saving and highly sensitive way for the application of SERS technology for the detection of antibiotic residues in real meat samples.