Contamination of food through the presence of pathogens can pose a serious risk to health. For safe food consumption, monitoring for the presence of pathogens is critical to identify and regulate microbiological contamination. Milk is no exception and requires rapid analysis of samples to identify unsafe bacteria levels. In this work, an aptasensor based on a quartz crystal microbalance with dissipation monitoring was developed to detect and quantify Staphylococcus aureus directly in whole UHT cow’s milk. The aptasensor demonstrated high sensitivity and was able to detect S. aureus in milk with a 33 CFU/mL limit of detection. Analysis was successful in milk due to the antifouling properties of the device, which is based on surface modification by 3-dithiothreitol propanoic acid (DTT_COOH), a novel antifouling thiol linker. Compared to bare and modified (dithiothreitol (DTT), 11-mercaptoundecanoic acid (MUA), and 1-undecanethiol (UDT)) QCM discs, the aptasensor’s antifouling in milk improved by about 82-96%. The antifouling QCM-based aptansensor’s excellent sensitivity and ability to detect and quantify S. aureus in whole UHT cow’s milk demonstrates that the system is applicable for rapid and efficient analysis of milk safety.