As an endocrine disruptor, bisphenol A (BPA) has many adverse effects on environmental safety and human health. Aiming at the existing problems at the current detection methods, herein, a 3D metal‐organic framework (Ni‐MOF) was fabricated as a carrier to achieve BPA aptamers immobilization inside its array channels under Mg2+ regulation. Based on the reversible quenching of 6‐FAM (6‐carboxyfluorescein) fluorescence labeled on the BPA aptamers during the encapsulation process, a fluorescent aptamer sensing platform was constructed for the quantitative detection of BPA in water. Morphology analysis, VASP (Vienna ab‐initio simulation package) and other kinetic simulations were performed to elaborate the mechanism of the influence of pore size, medium ions, etc. on the recognition process. Due to the in‐hole fixation strategy of aptamers, Ni‐MOF played an obvious protection aptamers. Not only the activity of BPA aptamer@MOF composite was maintained more than 50 % in complex environments such as pH 3.0–11.0, 30–70 °C and organic solvents, but also the aptamer was protected from nuclease hydrolysis under physiological conditions. The stability and application range of the sensor are greatly improved, detection limit of 0.34 μM. The result was expected to provide theoretical guidance for the rapid detection of pollutants in complex environments.