Different methods have been used to detect milk adulteration, but in recent years the use of nanomaterials has been introduced as an interesting tool, due to their optical properties. A gold nanoparticle (AuNP) probe application was developed in order to evaluate milk adulteration. The methodology relies on the colorimetric differentiation and the participation profiles of the hybridization DNA sequence with the AuNPs. Various concentrations, from 0.01 (traces) to 50%, of cows’ milk in goats’ milk samples were prepared for DNA extraction, further identification with the AuNPs and comparison with a conventional PCR. Also, a total of 40 dairy products from goat milk, commonly consumed in Greece, were tested. Negative and goat reaction mixtures showed a purplish coloured solution with a peak at > 570 nm, while samples containing bovine DNA had an absorbance closer to the characteristic peak of the AuNPs at 520-525 nm. Presence of bovine milk was detected even at traces level, achieving a detection level comparable to those achieved by conventional PCR. The use of AuNPs in milk products, provides a low-cost and easy-to-perform method and offers the possibility to detect fraudulent practices in various food matrices.