Effect of sulfuric acid (SA) on the heat treatment of electrospun polyacrylonitrile (PAN) fibers and their application on guanine oxidation detection in single-strand DNA molecules (ssDNA) were studied. In this regard, two different approaches were performed. Aqueous sulfuric acid solutions were poured on the pure PAN nanofibers and heat treatment process was performed to determine the maximum temperature at which fiber structures does not deteriorate. Also, SA was directly added into electrospinning solutions with different ratios and heat treatment process was applied to the as-spun PAN/SA fibers. The fibers were produced both directly on a grounded plate and cylindrical pencil graphite surfaces (PGE) for the fiber characterizations and biosensor measurements. Single strand DNA (ssDNA) molecules were immobilized on the samples for the electrochemical investigation of guanine oxidation signal. 0.971 and 3.69 mu A guanine oxidation signal intensities were detected for neat PGE and heat-treated PAN/SA fiber coated PGE. The formation of sulfonic groups on the fibers has driven more ssDNA attachment on the surface, and hence increased the guanine oxidation signal intensity dramatically (about 261.4% increment). The results have shown promising future of heat-treated PAN/SA micro/nanofibers for sensitive detection of guanine oxidation signal from genetic molecules.