Abstract: In this study, iron (Fe) and cobalt (Co) co-doped zirconium-based metal-organic framework nanomaterials (Fe, Co co-doped UiO-66) were applied to acetylcholinesterase biosensors to detect organophosphorus compounds. By constructing an enzyme biosensor based on Fe, Co co-doped UiO-66, this study aimed to enhance its detection sensitivity and selectivity for organophosphorus compounds, while exploring its potential applications in pesticide residue monitoring. The synthesized material exhibited a highly dispersed octahedral structure, with Fe- and Co-ion doping maintaining the original crystal configuration. Experimental verification confirmed the suitability of this material for the fabrication of biosensors. The following optimal detection conditions were established: Material dosage of 2 mg/mL, enzyme loading of 0.6 U, phosphate buffered saline buffer pH7.5, and incubation time of 20 min. Scan rate studies revealed that electrode operation was controlled by surface processes. The developed biosensor displayed a linear response range of 1×10⁻¹⁴ mol/L to 1×10⁻⁸ mol/L for organophosphorus compounds, with a detection limit as low as 2.3×10⁻¹⁵ mol/L. Spiked recovery tests on tomato and mango samples showed recovery rates of 103.21%~106.16% and 99.12%~103.31% respectively, with relative standard deviations of 3.44%~4.29% and 3.63%~6.73%, thereby demonstrating the reliability of the sensor and its effectiveness in practical applications. Benefiting from its excellent electron conductivity and abundant active sites, the material significantly enhanced acetylcholinesterase immobilization efficiency, thereby providing a novel strategy for developing high-performance pesticide residue detection technologies.