Advances in many aptamer-based applications will require a better understanding of how an aptamer's molecular recognition ability is affected by its incorporation into a suitable matrix. In this study, we investigated whether a model aptamer system, the sulforhodamine B aptamer, would retain its binding ability while embedded in a multilayer polyelectrolyte film. Thin films consisting of poly(diallyldimethylammonium chloride) as the polycation and both poly(sodium 4-styrene-sulfonate) and the aptamer as the polyanions were deposited by the layer-by-layer approach and were compared to films prepared using calf thymus DNA or a random single-stranded oligonucleotide. Data from UV-vis spectroscopy, quartz crystal microbalance studies, confocal microscopy, and time of flight secondary ion mass spectrometry confirm that the aptamer's recognition of its target is retained, with no loss of specificity and only a modest reduction of binding affinity, while it is incorporated within the thin film. These findings open up a raft of new opportunities for the development and application of aptamer-based functional thin films.