Using radix pueraria flavonoids (RPFs) as a reducing and stabilizing agent, we report a simple, cost-effective, and ecologically friendly green synthesis technique for gold nanoparticles (AuNPs) in the present study. Ultraviolet–visible (UV) spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared (FTIR), and X-ray diffraction (XRD) investigations were used to characterize the AuNPs. The results demonstrated that the produced AuNPs were nearly spherical and that their particle sizes had a mean diameter of 4.85 ± 0.75 nm. The “Green” AuNPs, exhibiting remarkable peroxidase-like activity and Michaelis–Menten kinetics with high affinity for H 2O 2 and 3,3′,5,5′-tetramethylbenzidine (TMB), were effectively applied to the fabrication of a sensitive nonenzymatic enhanced electrochemical sensor for the detection of cholesterol (Cho). Under optimum circumstances, it was possible to establish two linear ranges of 1–100 and 250–5000 μmol/L with a detection limit of 0.259 μmol/L (signal/noise ratio (S/N) = 3). The suggested sensor was utilized with satisfactory findings to determine the amount of Cho in food samples.