In this study, glucose oxidase (GOX) was immobilized in electrospun polylactide (PLA) fibers 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 system in model solutions using GOX-in-PLA fibers provided a more sustained generation of antimicrobial OSCN- than direct activation using H2O 2. Preliminary evaluation on milk samples showed that the electrospun GOX-in-PLA microfibers are capable of activating the naturally present LP system. Microbial analysis of LP system activated in milk exerted antimicrobial effect, indicating that they may be promising for active food packaging applications to extend the shelf life of milk.