High-precision and miniature biosensors are in urgent demand with respect to the organophosphorus pesticides used extensively in agriculture, especially for Chlorpyrifos. In this study, the novel CuFe 2 O 4 -Graphene Quantum Dots (CuFe 2 O 4 / GQDs) Magnetic Nano-Particles (MNPs) was prepared to amplify color signal as peroxidase mimetic using one-step hydrothermal method with electrostatic adsorption. Further combined with well-designed microfluidic chip containing enzyme inhibition reaction, color reaction and uV spectrophotometric detection areas, a colorimetric biosensor was developed based on CuFe 2 O 4 /GQDs for Chlorpyrifos determination. Experimental results of the biosensor displayed a linear range from 3.2 × 10 -8 M to 6 × 10 -7 M with a correlation coefficient of 0.99258 under the optimized conditions, and the Limit of Detection (LOD) of 1.07 × 10 -8 M based on S/N = 3 and the sensitivity of 855.26 (absorbance) per 1 mM. Great advantages have been exhibted in the Microfluidic colorimetric biosensor due to the small volume, and high precision for determination of low concentrations of organophosphorus pesticides, largely simplifying the tedious operation and lowing LOD, which is great benefit to the further development of low-cost, portable and high-precision biosensors.