Biotin has been incorporated into polylactic acid (PLA) nanofibers by electrospinning polymer solutions (22 wt% PLA) with different biotin concentration in dimethyl formamide at elevated temperature. Scanning electron microscopy indicated uniform morphology and small diameters for the fibers containing 0, 3, 10 and 18 % of biotin (w/w PLA). Fiber diameters were insignificantly influenced by biotin incorporation. The membranes were characterized by electron probe microanalysis (EPMA) and found a linear correlation between the biotin concentration in the initial dopes and that in the resulting membranes. EPMA mapping indicated the successful incorporation of biotin in nanofibers and homogeneous distribution of biotin on the membranes at different concentrations. The surface-available biotin in the nanofibrous membrane containing 18 % biotin was determined using HABA/streptavidin competitive assay, the stoichiometric displacement of HABA from the HABA/streptavidin complex by biotin due to the much higher affinity of biotin for the protein. The assay indicated that 17.7 mg biotin/g membrane, or 9.8 % of the total amount of the incorporated biotin was available to binding with streptavidin for this electrospun PLA/biotin membrane. The surface-available biotin can serve as the attachment sites for immobilization of streptavidin which can in turn further immobilize a wide variety of relevant biotinylated biorecognition elements, making such electrospun membranes ideal candidate substrates in biosensing applications.