<p> A new kind of electrochemical biosensor was fabricated by modifying a glassy carbon electrode (GCE) with Ni-doped ZnO nanorods suspension for the sensitive and selective detection of miRNA-21. The electrode surface was coated with polydopamine (PDA), crosslinked with glutaraldehyde, and functionalized with DNA probes. Bovine serum albumin (BSA) was then used to block non-specific binding sites. Electrochemical analyses were adopted to systematically investigate the progress of the stepwise electrode modification and the hybridization. The electrochemical characterization indicated a gradual decrease in current response and an increase in charge-transfer resistance after each modification step, verifying the successful assembly of the sensing interface. Following optimization, the biosensor could detect concentrations as low as 21.30 fM with a dynamic linear range of 10^− 6 nM to 10⁵ nM, good reproducibility, and selectivity to the mismatch and non-complementary sequences. Crucially, recovery experiments in spiked human serum samples gave satisfactory results, confirming the clinical potential of the system. In general, the Ni-doped ZnO nanorod-based biosensor provides sensitive, label-free, and low-cost detection of circulating miRNAs. Its modularity allows for simple conversion for detection of other nucleic acid-based biomarkers, implicating it as a useful diagnostic tool for early cancer diagnosis and point-of-care screening.</p> Graphical abstract <p></p>