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A POP-based electrochemical aptasensor has been successfully designed and fabricated, which has outstanding physicochemical stability, reproducibility and reproducibility. Meanwhile, this fabricated aptasensor has ultrahigh selectivity and out-bound sensitivity toward AMP even in various real samples. • An exceptionally stable POP-based electrochemical aptasensor was successfully fabricated and used to detect ampicillin. • This biosensor exhibits the lowest LOD of 1.33 fg·mL–1 (3.30 fM). • This aptasensor has the ability to precisely identify ampicillin in various real samples. A novel porous organic polymer (POP) was designed and prepared by the coupled polymerization of 1, 3, 6, 8-Tetraphenylpyrene and α , α ′-dibromo- p -xylen. The as-synthesized POP is an extended π -conjugation framework with high surface area, excellent stability and large pore cavity, thus bestowing the outstanding carrying capacity of aptamer strands through π-π stacking interaction to rationally fabricate a ultrasensitive electrochemical aptasensor. The biosensor exhibits the lowest limit of detection of 1.33 fg·mL–1 (3.30 fM) for detecting trace ampicillin (AMP) by using electrochemical impedance spectroscopy. Furthermore, this biosensor can accurately monitor AMP in multiple real samples. This work provides the greatly potential applications of POPs in the fabrication of electrochemical biosensors for sensitively detecting antibiotics or other analytes. [ABSTRACT FROM AUTHOR]