Despite recent advances in signal reporter-based assays for bacteria detection and profiling, the low-cost, ultrasensitive, accurate, and fast diagnosis remains a challenge for better patient care. Herein, we present a novel bacteria identification method based on α-hemolysin-labeled sandwich assay (HLSA). A pore-forming protein, α-hemolysin, is used as an electrical signal reporter. The assay takes advantage of the specific binding of target nucleic acid with two hybridization probes: capture probe decorated magnetic microparticles and oligonucleotides detecting probe and α-hemolysin modified gold nanoparticles. α-Hemolysin was then released by competitive gold binding peptide incubation into an electric cell with a lipid bilayer between the electrodes. The nanopores formed by α-hemolysin on the lipid layer allowed target nucleic acid concentration-dependent currents for quantification. Sandwich probes against 16S rRNAs of 10 common bacteria pathogens were designed and single cell level nucleic acid concentration detection was achieved. Compared with nanopore technique-based DNA sequencing, HLSA gives a quantitive and straightforward readout that is not dependent on an ultrasensitive and expensive instrument (Axopatch 200B amplifier), thus, is faster and requires no large-scale instruments. Also, since α-hemolysin-modified nanoparticles will be washed out before the α-hemolysin releasing step without the target nucleic acid, no current will be detected, and thus, the assay is more specific. The current strategy based on the electrical signal reporter offers a new insight for pathogen and virus diagnostics.