Gentamicin (GEN) is an aminoglycoside antibiotic widely used in animal husbandry because of its low cost and high therapeutic efficacy; however, its residues in milk pose a potential risk to food safety. In this study, a truncated aptamer (G41) with high affinity toward GEN was obtained through molecular docking and molecular dynamics simulations. Gold nanoclusters (Au NCs) were synthesized using an l-proline reduction method and conjugated with G41 via Au-S bonds to form Au NCs-Apt. Meanwhile, amino-functionalized hollow mesoporous silica (HMSN-NH2) was employed as a carrier to load rhodamine 6G (R6G), with Au NCs-Apt acting as a gating molecule to encapsulate R6G through electrostatic interactions.In the presence of GEN, the specific binding between GEN and G41 induced the dissociation of Au NCs-Apt from the surface of HMSN-NH2, resulting in the release of R6G and a significant enhancement of the fluorescence signal at 556 nm (I556), while the fluorescence signal of Au NCs at 442 nm (I442) remained essentially unchanged. The proposed sensor exhibited an excellent linear response over the concentration range of 20-4000 ng/mL (R2 = 0.9977) with a low detection limit of 2.36 ng/mL. Moreover, The aptasensor showed good repeatability, with relative standard deviations (RSD) below 5% based on three independent measurements (n = 3). In addition, the method was applied to the determination of GEN in spiked milk samples, affording recoveries in the range of 98.35-101.49%, demonstrating its applicability to complex sample matrices. By integrating HMSN with a ratiometric fluorescence aptasensing strategy, this study provides a highly stable and anti-interference analytical platform for the monitoring of antibiotic residues in food.