A qualitative real-time PCR-based method for the detection of the Brazil nut (Bertholletia excelsa) component in food is described. The method consists of DNA isolation by chaotropic SPE and the subsequent PCR with Brazil nut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the gene encoding for the 11s globulin of the Brazil nut. The method was positive for eight Brazil nut samples from the market and negative for all other tested plant and meat materials used in the food industry (36 samples). The intrinsic LOD of the method was 10 pg Brazil nut DNA. Using a series of model nut paste mixtures with defined Brazil nut contents, a practical detection limit of 0.1% (w/w) Brazil nut was estimated. Practical applicability of the entire method was tested by qualitative analysis of nine food samples; no discrepancies between the declared and detected Brazil nut contents were found. The presented PCR-based method is useful for sensitive and selective detection of Brazil nut in food samples.