This study employs a microfluidic system as an in vitro tool to simulate the human physiological environment and to explore its feasibility for the development of lead compounds for Alzheimer’s disease. Through the use of microfluidics, the study aims to reduce sample consumption and shorten experimental time, thereby replacing part of animal testing. First, the extraction efficiency of immersion and microfluidic methods was compared, and the results showed that the microfluidic extraction achieved the best performance and the fastest reaction time under identical sample conditions. Subsequently, colorimetric assays (DTNB and 3-NPH HCl) were conducted, followed by quantitative analysis using an ELISA reader and HPLC as the basis for microfluidic analysis, which was further applied to the acetylcholinesterase (AChE) inhibition assay. Furthermore, cypermethrin and its degradation products were used as examples to investigate their effects on AChE activity and the viability of SH-SY5Y neuroblastoma cells. The results indicated that cypermethrin exhibited higher enzyme inhibition and showed no significant cytotoxicity at concentrations ≤ 200 μM. Moreover, the molecular docking results were consistent with the experimental findings. In conclusion, the microfluidic system enhances reaction rates and extraction efficiency, and future studies will integrate cell culture applications for the development of lead compounds targeting Alzheimer’s disease.