Traditional microbiology analysis is usually hindered by the long time-cost and lack of portability in many urgent situations. In this work, we developed a novel electrochemical DNA biosensor (E-biosensor) for sensitive analysis of the 16S rRNA gene of five bacteria, using a consecutive adenine (polyA) probe. The polyA probe consists of a polyA tail and a recognition part. The polyA tail can combine onto the gold surface with improved controllability of the surface density, by conveniently changing the length of polyA. The recognition part of the capture probe together with two biotin-labeled reporter probes hybridize with the target DNA and form a stable DNA-tetramer sandwich structure, and then avidin-HRP enzyme was added to produce a redox current signal for the following electrochemical detection. Finally, we realized sensitive quantification of artificial target DNA with a limit of detection (LOD) of 10 fM, and excellent selectivity and reusability were also demonstrated. Importantly, the detection capability was equally good when facing bacterial genomic DNA, due to the base-stacking force of our multireporter-probe system, which can help to break the second structure and stabilize the probe-target complexes. Our biosensor was constructed on a 16-channel electrode chip without any polymerase chain reaction (PCR) process needed, which took a significant step toward a portable bacteria biosensor.