Enzyme-inorganic hybrid nanoflowers (HNFs) have shown excellent sensing capabilities due to their large specific surface area as well as the simplicity and mildness of the preparation process. However, coupling HNFs to electrodes to fabricate a uniform and controllable enzymatic electrochemical sensing interface remains a challenge. Here, we proposed an aptamer-induced in-situ fabrication strategy for preparing an HNF-based electrochemical sensor with ideal performance. Central to this strategy is the introduction of acetylcholinesterase (AChE)-specific binding aptamer (Apt), which induces the in-situ growth of AChE-copper phosphate (Cu₃(PO₄)₂) HNFs on the surface of carbon paper (CP). In addition, a dense gold nanoparticle (AuNP) layer was electrodeposited on the CP for anchoring Apt and further extending the electroactive surface area. The prepared AChE-Cu₃(PO₄)₂ HNF/Apt/AuNP/CP biosensor exhibited a wide detection range from 1 to 10⁷ pM for the four organophosphorus inhibitors, including isocarbophos, dichlorvos, methamidophos, and parathion, with detection limits down to 0.016, 0.028, 0.071, and 0.113 pM, respectively. With the reactivation of pralidoxime chloride, the electrode can still recover 98.1% of the response after five times of repeated use. In real sample detection, the biosensor achieved high recoveries from 96.45% to 100.13%. The detection target may be extendable to other AChE inhibitors (e.g., drugs for Alzheimer’s disease). This study demonstrates for the first time the feasibility of using aptamers as an inducer to fabricate an electrochemical enzyme sensing interface in-situ. This strategy can be used to fabricate other enzyme-based biosensors and therefore has broader applications.