MicroRNA (miRNA), a large and growing class of 18–24-nucleotide long, noncoding RNA molecules in all known animal and plant genomes, is a key player in gene regulation. The functions of miRNA are yet to be understood with respect to how and where it is produced and the changes within an organism associated with variations in miRNA expression level. The expression profiles serve as molecular diagnostics for diseases and new targets in drug discovery. Consequently, highly sensitive and selective detection of miRNA is playing a significant role in understanding miRNA functions. Existing major methods of detecting miRNA are dependent on hybridization, in which a target miRNA molecule is hybridized to a complementary probe molecule. Recently developed detection methods introduce nanomaterials to the hybridized duplex to enhance the sensitivity. However, all of them are indirect, involving labeling or conjugating process. To overcome the above-mentioned issues, we have demonstrated a highly sensitive and label-free direct detection method for miRNA by using peptide nucleic acids (PNAs)-functionalized silicon nanowires (SiNWs) biosensor. The sensor is capable of detecting target miRNA as low as 1 fM (10–15 M), as well as identifying fully matched versus mismatched miRNA sequences. More importantly, the SiNW biosensor enables miRNA detection in total RNA extracted from HeLa cells. The developed detection method shows potential applications in label-free, early detection of miRNA as a biomarker in cancer diagnostics with very high sensitivity and good specificity.