Developing efficient electrochemical aptasensors is one of the effective methods to address the thorny issue of antibiotic abuse that poses a threat to human health. Functional materials are crucial for improving detection performance. Combining the advantages of gold (Au) nanoparticles, metal–organic frameworks (MOFs), and covalent organic frameworks (COFs), a multivariate Au@MOF@COF nanocomposite was designed and synthesized to immobilize aptamers for electrochemical aptasensing of trace oxytetracycline (1.0 × 10⁻⁴–0.1 ng·mL⁻¹) with great sensitivity, good repeatability, fine stability, and remarkable selectivity. Especially, quantitative detection and analysis of oxytetracycline can be achieved in real milk samples using this aptasensor.