In this letter, the facial noncovalent adsorption of single-stranded DNA (ssDNA) provided single-walled carbon nanotubes (SWNTs) with biofunctionality while their superior properties were retained. In this case, we innovatively demonstrated the feasibility of employing the negative surface charge of ssDNA-SWNTs to realize layer-by-layer electrostatic self-assembly. On the basis of such a sandwichlike structure, an applicable glucose microbiosensor with direct electrochemistry and high performance was fabricated. The proposed protocol provided an ideal platform for various sensing applications, and might have profound influence on related nanotechnology.