The application of nanotechnology in biomedical field has enormous potential in basic and applied research. Micro or nanofibers produced by electrospinning technique offer excellent properties because of large specific surface area, high porosity, and ability to incorporate functional additives. Here we embedded biotinylated bovine serum albumin into polylactic acid (PLA)-polyethylene glycol (PEG) fibers, which enabled specific immobilization of fluorescently labelled avidin. An alkaline phosphatase enzyme was immobilized via biotin-streptavidin interaction on the hybrid nanofibers, demonstrating the suitability of the material for biosensing applications. These functional nanofibers provide a promising platform for development of biosensors and other biofunctional materials utilizing avidin-biotin as a generic and robust immobilization method. (c) 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 356-362, 2017.