This paper describes a disposable amperometric biosensor for simple and sensitive determination of miRNA-21. The bioplatform consists of gold nanoparticles-nanostructured electrode surfaces on which a direct hybridization assay involving the immobilization of a specific thiolated DNA capture probe and recognition of the formed DNA-miRNA-21 heteroduplex by a specific antibody is implemented. The antibody is further recognized through its Fc region by a commercial bacterial protein containing 40 units of horseradish peroxidase (HRP) (ProtA-polyHRP40). The amperometric detection of the hybridization process using the HO/hydroquinone system allows quantification of the target miRNA in the 0.096-25 pM linear range with a detection limit of 29 fM (0.29 amol in 10 μL of sample). The bioplatform offers excellent selectivity against noncomplementary sequences and very acceptable against sequences with an unpaired base (only 30% of the response obtained for the target miRNA). In addition, the bioplatform was shown to be useful for the determination of the endogenous content of the target oncomiR directly in blood serum from breast cancer patients and in breast cancer cells using only 10 ng of total extracted RNA.