Nonspecific binding of proteins from complex food matrices is a significant challenge associated with a biosensor using gold nanoparticles (AuNPs). To overcome this, we developed an efficient EDTA chelating treatment to denature milk proteins and prevent their adsorption on AuNPs. The use of EDTA to solubilize proteins enabled a sensitive label-free apta-sensor platform for colorimetric detection of Staphylococcus aureus in milk and infant formula. In the assay, S. aureus depleted aptamers from the test solution, and the reduction of aptamers enabled aggregation of AuNPs upon salt addition, a process characterized by a color change from red to purple. Under optimized conditions, S. aureus could be visually detected within 30 min with the detection limit of 7.5 × 10 4 CFU/mL and 8.4 × 10 4 CFU/mL in milk and infant formula, respectively. The EDTA treatment provides new opportunities for monitoring milk contamination and may prove valuable for biosensor point-of-need applications.