This study aimed to propose a novel surface-enhanced Raman spectroscopy (SERS)-based aptasensor by using 4-mercaptobenzoic acid (4-MBA) modified gold@silver core-shell nanoparticles (Au@Ag NPs) for detecting kanamycin for the first time with outstanding sensitivity and selectivity in the real milk samples. To achieve a high SERS activity substrate, the Au@Ag NPs with uniform shapes in a diameter of 32.2 ± 3.4 nm were synthesized successfully by seed-mediated growth and then modified with the Raman reporter 4-MBA. Under the optimal conditions, this aptamer sensing system exhibited a linear range between 6.67 × 10^− 10 g/mL and 2 × 10^− 7 g/mL with the limit of detection (142 pg/mL) of kanamycin. Moreover, the KANA-specific aptamer endowed a good specificity to this biosensor for the negative interference from other analogues (streptomycin sulfate, gentamicin sulfate, and penicillin). Finally, the aptasensor was applied in the artificially contaminated milk (1–100 ng/mL) samples and compared with the HPLC-MS technique, with the result indicating great practicality of the aptasensor. This paper has provided a potential aptasensor for simple and rapid trace-sensing of the harmful antibiotic residue in milk.