Electrospun water-soluble nanofibers as enzyme supports have potential to overcome limitations of traditional immobilization by increasing the surface area-to-volume ratio, reducing diffusional limitations, and improving storage stability. In this study, p-galactosidase (lactase, Aspergillus oryzae) was electro spun into polyethylene oxide (PEO) nanofibers with Pluronic F-127 (F127) (polyethylene oxide/polypropylene oxide block copolymer) to enable dry storage stability and subsequent rapid dissolution upon use in applications such as sensing, bioactive packaging, and bioprocessing. Nanofibers were cylindrical (average diameter similar to 300 nm) and exhibited no beading after spinning or storage. After electrospinning, enzymes retained up to 92% of their native activity. After 4 weeks storage, the immobilized enzyme fiber retained up to 44% of the activity of the original fiber. These results suggest that co-spinning enzymes in a polymer nanofiber matrix can improve activity retention and dry storage stability compared to traditional immobilization methods and enables rapid dissolution for ease of use. (C) 2014 Elsevier B.V. All rights reserved.