The first label-free modified RNA-aptasensor for the detection of small molecules in biological samples is presented. A competitive displacement assay was applied to the detection of aminoglycoside neomycin B in whole milk using a fully 2'-O-methylated RNA aptamer with faradaic impedance spectroscopic (FIS) detection. Neomycin B in solution displaces the aptamer from its complex with the SAM-immobilized neomycin B. The reusable aptasensor is capable of discriminating neomycin B from paromomycin, which differs from it in the substitution of a single amine group with a hydroxyl one. We show that the modified endonuclease-resistant RNA aptamer maintains the exquisite selectivity of the natural aptamer and allows the examination of biological samples of high protein content. This detection scheme can be readily adapted to the detection of other aminoglycosides and small molecules.