This paper describes the characterization and optimization of a reusable DNA microsensor array for rapid biological agent detection developed in previous publications. (Fig.1) [1–3] This MEMS based DNA sensor utilizes a standard three-electrode electrochemical cell configuration with novel micro fabricated structure design to minimize non-specific binding. The sensor module is easily to be adapted to various protocols and can be used for rapid detection of macromolecules (DNA, RNA) from targets such as uropathogenic Escherichia coli (E. coli) in urine and microorganisms causing otitis media (middle ear infection). Less than 105 E. coli cells can be detected from the urine sample of a patient with urine tract infection. The sensitivity is enhanced by appropriate sensor characterization and surface modification. The total detection time including sample preparation can be reduced to 25 minutes by using a POD conjugated oligonucleotide.