Portable, easy-to-use and cost-effective sensing devices are desirable in healthcare, environmental monitoring and food safety. Herein we employ polyelectrolyte-aptamer (PE-aptamer) multilayered films that exhibit target-responsive permeability for colorimetric and electrochemical sensing. We present the quantitative detection of an exemplary small molecule, quinine, and address the potential for detection in complex media by examining interference effects. We optimize the film composition and investigate the importance of the structural-switching ability of the aptamer. The results from both platforms are corroborated to provide an outlook on the applicability of the PE-aptamer film for sensing. The label-free detection combined with the readily adaptive assembly process could be invaluable for diverse analytical fields.