Enrofloxacin (ENR), a third-generation fluoroquinolone veterinary antibiotic, is widely used in aquaculture and animal husbandry for bacterial infection control due to its broad-spectrum antimicrobial activity, potent bactericidal efficacy, and cost-effectiveness. However, its excessive and non-standard use has caused widespread residues in animal-derived foods, which pose severe risks to human gastrointestinal, nervous and hepatorenal systems, induce bacterial resistance, and threaten global food safety and ecological sustainability. Herein, we developed a label-free, rapid, and highly sensitive fluorescence aptasensor for ENR detection based on G-triplex signal amplification. The ENR-specific aptamer was pre-hybridized with a guanine-rich sequence to suppress G-triplex formation and maintain a low thioflavin T (ThT) background. Upon ENR addition, target binding releases the guanine-rich strand, which folds into a stable G-triplex and strongly enhances ThT fluorescence for quantitative detection. Under optimal conditions, this aptasensor showed a linear range of 1.0 nM to 1.0 μM (R = 0.9925) with a detection limit of 1.2 nM. The assay takes only 25 min without complex instruments or chemical labeling. It exhibited excellent selectivity and satisfactory recoveries (92.36-105.78%) in spiked milk samples, providing a facile, low-cost, and reliable tool for on-site ENR residue screening in complex food matrices.