This study focuses on the application of a new electrode configuration for biosensing. An electrochemical DNA biosensor was developed based on the recognition of target DNA by hybridization detection with immobilized capture synthesized 15-mer single-stranded deoxyribonucleic acid (ssDNA) capture probe on a chemically modified carbon fiber microelectrode (CFME). The capture probe was covalently attached to amine-labeled ssDNA using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosulfosuccinimide (NHS) modified CFME. Square wave voltammetry (SWV) was used to monitor the hybridization reaction on the capture probe electrode. The covalent immobilized capture probe could selectively hybridize with its target DNA to form double-stranded DNA (dsDNA) on CFME surface by using methylene blue (MB) as a hybridization indicator. The decrease of the peak current of MB, an electroactive indicator, was observed upon hybridization of the probe with the target DNA. The peak current of MB was found to increase in the following order: hybrid-modified CFME, single-base mismatch hybrid-modified CFME, non-complementary modified CFME. The results show that the developed biosensor has a good calibration range between 0.5 and 10 ppm, with detection limit of 0.72 ppm.