Pine nuts are a known source of food allergens and several cases of adverse immunological reaction after ingestion have been reported. To protect allergic consumers, methods to unequivocally detect the presence of pine nuts in complex matrices must be developed. A Taqman-based real time PCR method for the detection of Pinus spp. was set up. A homemade pesto spiked at known concentration of pine nut powder was used as model food. Moreover, DNA was purified from commercial foods declaring or not the presence of pine nuts. The method displayed a very high efficiency and specificity for the genus Pinus. The intrinsic LOD was 1 pg of DNA, while the practical LOD evaluated on model foods was 0.1 ppm of pine nuts powder, the lowest ever registered for the detection of food allergens via real-time PCR. Finally, the declared presence/absence of pine nut in commercial foods was confirmed.