A simple and efficient way of dispersing hydrophobic nanofibers in aqueous solution was devised, and its utility in production and application of enzyme-loaded nanofibers was demonstrated. Polystyrene-based nanofibers were produced via an electro-spinning process. A small amount of maleic anhydride group in the polystyrene fiber was used for covalent attachment of lipase onto the fiber surface. The pristine polystyrene nanofibers are hydrophobic and aggregate in water, forming a tightly collapsed clump. These nanofibers can be dispersed in a surfactant-free aqueous solution via a simple alcohol pretreatment. The tightly aggregated electro-spun polystyrene nanofibers can be dispersed into a loosely entangled structure in aqueous alcohol solution. Once treated with aqueous alcohol solution, the polystyrene nanofibers remain dispersed even in DI water as long as the nanofibers are not dried during the washing step. The dispersion of polystyrene nanofibers increases the enzyme loading up to similar to 8 times and augments the steady-state conversion of a continuous flow reactor filled with enzyme-loaded nanofibers.