In the present manuscript, a modified glassy carbon electrode with BSA templated Au–Ag bimetallic nanoclusters (Au–Ag@BSA/GCE) was employed for the rapid, selective and sensitive determination of methyl parathion (MP) as an enzyme-less electrochemical biosensor. The properties and morphology of the synthesized bimetallic nanoclusters were characterized by fluorescence, transmission electronic microscopy (TEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy techniques. Additionally, the properties of the prepared biosensors were investigated by cyclic voltammetry (CV), square wave anodic stripping voltammetry (SWASV) and electrochemical impedance spectroscopy (EIS). The results show that Au–Ag@BSA/GCE greatly catalyzed the redox reaction of MP during electrochemical detection. Under optimal conditions, the linear dynamic ranges of MP were obtained between 0.02 to 8.0 and 8.0 to 200 mmol L1, with a detection limit of 8.2 nmol L1. The selectivity, stability, repeatability, and reproducibility of Au–Ag@BSA/GCE were studied, and the obtained data suggest that the proposed biosensor has an excellent capacity as an alternative to enzyme inhibition-based biosensors for the determination of MP. The constructed biosensor was able to successfully electrochemically determine the presence of MP in different water, apple, vegetable, and soil samples.