Enzyme-functionalized solution-blown nonwoven (EFSBN) fibers were produced by a single-step solution blow spinning method that utilizes high-velocity gas to simultaneously extrude the codissolved polymer–solvent–enzyme spinning solution and evaporate solvent at mild conditions to form a nanofibrous web with preserved enzyme activity. A broad concentration range of 0.6–7.4 wt % protein of subtilisin A protease from Bacillus licheniformis was successfully entrapped by poly(ethylene oxide) (PEO) during solution blow spinning nonwoven web production. The presence of enzyme protein in the solid nanofibers was detected by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Time of flight-secondary ion mass spectroscopy and laser confocal microscopy revealed the immobilized enzymes were mainly positioned inside the fibers and homogeneously distributed throughout the webs. Scanning electron microscopy showed that the fiber shape and diameter of PEO nanofibers containing enzymes were irregular compared to PEO-only nanofibers. Residual enzyme activity in the webs was measured by redissolving fibers in buffer and comparing the released enzyme activity to nonimmobilized free enzyme using a casein substrate-based assay. Immobilized protease (1.3% (w/w) protein in solid dry nanofiber webs) retained more than 90% of the free enzyme activity. Protease immobilized in solid nanofiber webs exhibited long storage stability at ambient (∼22 °C) and 4 °C temperature storage conditions, with more than 60% remaining catalytic activity after 300 days compared to the initial activity. Immobilized protease had equally good thermal stability as a stabilized liquid commercial protease, both retaining above 95% of their initial activity after treatment for 12 h at 65 °C. In contrast, the same liquid protease diluted in buffer lost activity within 2 h at that temperature. The nondusting, readily aqueous-soluble EFSBN solid materials are easy to handle and have good storage stability compared to liquid products.