A paper-based DNA biosensor was developed for food authenticity testing using dairy products as a model. DNA from milk-based samples was isolated, and species-specific DNA sequences were amplified and identified by the biosensor using specific DNA probes. The properties of gold nanoparticles were exploited for visual detection. The biosensor was applied for detection of three species, namely cow, sheep and goat, while as low as 1.6 fmol for cow and goat, and 3.1 fmol for sheep PCR product were detected. Moreover, adulteration down to 0.01% could be detected, based on binary mixtures of cows', ewes' and goats' milk yogurt, containing 0.01 to 5% of cows' yogurt in ewes' and goats' yogurts, respectively. The proposed paper-based DNA biosensor offered 10 times higher detectability than other methods, good specificity and reproducibility, and could be applied easily for the detection of other adulterated food products, such as meat, olive oil and legumes.