The aims of this study were to encapsulate water-soluble bioactive agents into biodegradable hydrophobic polymers via emulsion electrospinning for drug delivery and tissue engineering applications and propose a simple and facile method to evaluate the bioactivity of the encapsulated protein. Proteinase K was selected as a model protein to be incorporated into poly(ethylene glycol)-poly(l-lactide) (PELA) ultrafine fibers. Core-shell structured fibers with single core or multi-core were observed. In vitro release study showed that after a burst release at the early stage, a sustained release was achieved, indicating that proteinase K was incorporated inside ultrathin fibers successfully. Results of in vitro incubation in Tris-HCl buffer at pH 8.6 and 37 A degrees C revealed that electrospun PELA membranes containing proteinase K (PELA-P) showed obvious morphological changes, large mass loss, and slight decreases in melting temperature, melting enthalpy and relative molecular mass in 7 days. Additionally, a significant drop in pH value of the buffer after incubation of the PELA-P membrane was also observed. These findings clearly showed that encapsulation of water-soluble bioactive agents inside hydrophobic polymers could be achieved by emulsion electrospinning without compromising their bioactivity.