Aptamer-based trapping techniques are in general suitable to replace common antibody-based enrichment approaches. A time-consuming isolation or clean-up is often necessary during sample preparation, e.g. for the detection of spores. For the development of bioanalytical routine approaches, aptamers with a high affinity to B. cereus spores were applied for the establishment and validation of an aptamer-based trapping technique in milk with fat contents between 0.3 and 3.5%. Thereby, enrichment factors of up to sixfold were achieved. The combination of an aptamer-based enrichment by magnetic separation and the subsequent specific real-time PCR detection represents a reliable and rapid detection system.