A highly sensitive microRNA (miRNA) biosensor that employs ruthenium oxide nanoparticle (RuO(2) NP)-initiated polymerization of 3,3'-dimethoxybenzidine (DB) and miRNA-templated deposition of an insulating poly(3,3'-dimethoxybenzidine) (PDB) film is described in this work. The biosensor was made of a mixed monolayer of oligonucleotide capture probes (CPs) and 4-mercaptoaniline on a gold electrode. Following hybridization with a RuO(2) NP-tagged target miRNA, a mixture of DB/H(2)O(2) in pH 5.0 0.10 M acetate buffer was applied to the biosensor. The RuO(2) NPs serve as polymerization initiator/catalyst for the polymerization of DB. And the hybridized anionic miRNA strands and free CPs serve as templates, guiding the deposition of PDB. The amount of the deposited PDB and its insulating power directly correlated to the concentration of the target miRNA in solution. Electrochemical impedance spectroscopic tests showed that a linear charge-transfer resistance-concentration relationship from 6.0 fM to 2.0 pM was attained after 60 min of incubation in the DB/H(2)O(2) mixture. There was no cross-hybridization between pre-miRNA and mature miRNA and very little cross-hybridization among closely related miRNA family members even at single-base-mismatched levels. This impedance-based biosensor offers an attractive alternative for miRNA expression profiling and may enable the development of a portable multiplexing miRNA profiling system.