This research attempted to determine the practicality of the integration of electrospun polyaniline nanofiber as the main sensing component into interdigitated gold microelectrodes to develop a biosensor platform for sensitive, selective, and label-free detection the of Cyclooxygenase-2 (COX-2) biomarker from pure and human serum samples. COX-2 is an important enzyme in pain biomarkers, inflammation and cancer cell proliferation, so it is necessary to develop a reliable biosensor that can sensitively and objectively quantify COX-2 enzyme expression for clinical diagnosis. Polyaniline nanofibers were prepared at four different diameters using electrospinning performed at four different flow rates. The performance of the electrospun polyaniline nanofiber based biosensor was evaluated in comparison with a plain control biosensor using electrochemical impedance spectroscopy. Significant improvement was observed in the sensitivity of the electrospun polyaniline nanofiber based biosensor revealing the remarkable capability of electrospun polyaniline nanofiber in robust and rapid detection of the COX-2 biomarker. This improvement was attributed to the large specific surface area of electrospun polyaniline nanofiber as well as its highly porous structure which enhances size-matched confinement, transduction and signal strength, thus increasing the sensitivity of the biosensor significantly. The fabricated nanofiber based biosensor was able to detect the target antigen with concentrations as low as 0.01pg/ml and 10fg/ml in pure and human serum samples, respectively, as well as remarkable selectivity towards Human Serum Albumin suggesting the significant contribution of this nanofiber based platform to the enhanced strength and sensitivity in COX-2 analyte detection.