It is known that different diseases have characteristic biomarkers that are secreted very early on, even before the symptoms have developed. Before any kind of therapeutic approach can be used, it is necessary that such biomarkers be detected at a minimum concentration in the bodily fluids. Here, we report the fabrication of an interdigitated sensing device integrated with polyvinyl alcohol (PVA) nanofibers and carbon nanotubes (CNT) for the detection of an inflammatory biomarker, C-reactive protein (CRP). The limit of detection (LOD) was achieved in a range of 100 ng mL−1 and 1 fg mL−1 in both phosphate buffered saline (PBS) and human serum (hs). Furthermore, a significant change in the electrochemical impedance from 45% to 70% (hs) and 38% to 60% (PBS) over the loading range of CRP was achieved. The finite element analysis indicates that a non-redox charge transduction at the solid/liquid interface on the electrode surface is responsible for the enhanced sensitivity. Furthermore, the fabricated biosensor consists of a large electro-active surface area, along with better charge transfer characteristics that enabled improved specific binding with CRP. This was determined both experimentally and from the simulated electrochemical impedance of the PVA nanofiber patterned gold electrode.