Owning to the misuse of the antibiotics in animal husbandry and agriculture, it is highly urgent to determine the quantification of antibiotics in biological systems by the simple, sensitive, and fast method. In this work, a novel nanoarchitecture of Co-based metal-organic frameworks (Co-MOF) and terephthalonitrile-based covalent organic framework (TPN-COF) was synthesized (represented by Co-MOF@TPN-COF), followed by the exploitation as the bioplatform of non-label aptasensor for detecting the most frequently used β-lactam antibiotics, ampicillin (AMP). The new porous hybrid material of Co-MOF@TPN-COF was synthesized by adding the as-prepared TPN-COF into the Co-MOF preparation system. The multilayered Co-MOF@TPN-COF nanosheets exhibit a high specific surface area (52.64 m2 g−1), nitrogen-rich groups and excellent electrochemical activity. As a result, large amounts of aptamer strands can be bound over the Co-MOF@TPN-COF nanosheets owning to the strong π–π stacking and hydrogen bonds. When detecting AMP by the electrochemical impedance spectroscopy, the fabricated Co-MOF@TPN-COF-based aptasensor exhibits an ultra-low detection limit of 0.217 fg mL−1 within the AMP concentration from 1.0 fg mL−1 to 2.0 ng mL−1, which was superior to those previously reported in literatures. In addition, this proposed aptasensor also shows high selectivity, good reproducibility and stability, acceptable regenerability, and favorable applicability in human serum, river water and milk. Therefore, the proposed Co-MOF@TPN-COF-based aptasensor has a great promise to be applied as a powerful tool in the fields of food safety. •A novel 2D nanocomposite of Co-MOF and terephthalonitrile-based COF was synthesized.•Co-MOF@TPN-COF nanosheets with high specific surface area, nitrogen-rich groups and excellent electrochemical activity.•Co-MOF@TPN-COF-based aptasensor for ultra-sensitive detection of ampicillin.•Excellent selectivity, stability, reproducibility and applicability for detecting ampicillin.