In this study, we report an ultrasensitive western blotting method by introducing gold nanoparticles (AuNPs) onto modified polyvinylidene fluoride (PVDF) membranes via atom transfer radical polymerization (ATRP). AuNPs can prevent protein or peptide transfer across the PVDF membrane to reduce target loss. The results suggest that the improved western blotting method can be used to detect target biomarkers with a satisfactory limit of detection (LOD).