ABSTRACT: In this study, glucose oxidase (GOX) was immobilized in polylactide (PLA) fibers that were used to activate the lactoperoxidase (LP) system in milk. The GOX-containing microfibers were electrospun from emulsions prepared by dispersing aqueous GOX in PLA dissolved in a chloroform and N,N-dimethylformamide blend, using sorbitan monopalmitate as an emulsifier. The enzymatic activity of GOX-in-PLA fibers (1100 ± 400 nm diameter) was more than 19 times higher than that of the GOX-in-PLA membrane formed by direct casting, due to the larger surface area of the electrospun fibers. The activation of LP in model solutions using GOX-in-PLA fibers provided a more sustained generation of antimicrobial OSCN− than direct activation using H2O2. Preliminary evaluation on milk samples showed that the electrospun GOX-in-PLA microfibers are capable of activating the naturally present LP system, indicating that they may be promising for active food packaging applications to extend the shelf life of milk.