Technical Abstract: Poly(ethylene-co-glycidyl methacrylate) (PE-co-GMA) nanofibers with abundant active epoxy groups on surfaces were fabricated through a novel manufacturing process. The prepared PE-co-GMA nanofibers with different average diameters ranging from 100 to 400 nm were aminated by reacting the epoxy groups with 1,3-diaminopropane. The resulting aminated PE-co-GMA nanofibers were subsequently biotinylated and then successfully applied to immobilize streptavidin–horseradish peroxidase (HRP) conjugate via specific, strong and rapid binding of biotin and streptavidin. The streptavidin–HRP immobilized PE-co-GMA nanofibers showed high activity, efficiency, sensitivity as well as good reusability. The results demonstrated that PE-co-GMA nanofibers prepared could be a promising candidate as solid support materials for potential biosensor applications.