Sensitive detection of biomolecules is integral for biomarker screening and early diagnosis. Herein, surface-initiated reversible-addition-fragmentation-chain-transfer (SI-RAFT) polymerization is exploited as a novel amplification strategy for highly sensitive electrochemical biosensing of DNA. Briefly, thiol-terminated peptide nucleic acid (PNA) probes are first self-assembled onto a gold electrode for the specific capture of target-DNA fragments; the carboxyl-group-containing dithiobenzoate 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPAD) is then tethered to the hybridized PNA-DNA heteroduplexes by means of the well-established carboxylate-Zr4+-phosphate chemistry and serves as the chain-transfer agent (CTAs) for subsequent SI-RAFT polymerization, which is thermally initiated in the presence of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) as the free-radical initiator and ferrocenylmethyl methacrylate (FcMMA) as the monomer. Through SI-RAFT polymerization, one target-DNA fragment can be labeled by a large number of electroactive Fc tags, giving rise to significant amplification of the electrochemical signal. The SI-RAFT-polymerization-based strategy does not involve the use of natural enzymes or complex nanomaterials, offering the benefits of low cost and easy operation. Under optimal conditions, the electrochemical signal is linearly related to the logarithm of the concentration of target DNA over the range from 10 aM to 10 pM ( R2 = 0.997), with a detection limit down to 3.2 aM, which is much lower than those of other amplification-by-polymerization-based methods. By virtue of its easy operation, low cost, and high efficiency, the SI-RAFT-polymerization-based amplification strategy is believed to have great application prospects in the sensitive detection of biomolecules.