The present work demonstrates the use of mesoporous nanofibers for the enhanced analytical performance of electrochemical biosensor. By exploiting the phase separation property of the block copolymers, a simple three-step process was used to generate the porosity in the nanofibers. Here we present the effect of the porosity on the sensing ability of the electrospun PS-b-PMMA nanofibers. The functional groups present on the nanofiber surface were characterized using DPV. The nanofibers modified electrode showed a large decrease in the oxidation current with the increase in the pH from 4.2 to 6.8 for the anionic redox couple whereas the change in the current is negligible for a neutral redox couple, this suggested the presence of -COOH groups. A one-step process was used for the immobilization of biotin. There were about 35.5 % and 66.6 % decrease in the redox current for the as-spun and porous nanofibers after functionalization respectively which indicate the presence of a high amount of active sites in the porous nanofibers. Finally, the sensor response was studied using streptavidin (1μg/ml–1fg/ml) as a model analyte. CV studies showed a 2.7-fold increase whereas DPV showed a 6-fold increase in the sensitivity for the porous nanofibers as compared to the as-spun nanofibers. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim