Polyethylene glycol and polylactic acid bioactive membranes were developed for glucose sensor applications. Polylactic acid and polyethylene glycol were co-axially electrospun into a nonwoven, nanofibrous membrane composed of a core-sheath fiber structure. Differential scanning calorimetry and confocal microscopy were respectively utilized to confirm the polymer composition and fiber structure of the electrospun membrane. To create the bioactive membrane for glucose sensing, a bienzyme system and colorimetric agent were encapsulated in the polyethylene-glycol-polylactic-acid fiber structure. By encapsulating horseradish peroxidase and glucose oxidase in the polyethylene glycol core and o-dianisidine in the polylactic acid sheath of the fiber, immersion in glucose solutions causes the oxidized colorimetric agent to diffuse into the solution, allowing for colorimetric measurement with the glucose solution. UV-vis spectroscopy confirmed successful glucose sensing, showcasing a linear relationship in absorbance at 440 nm and mM concentration of glucose solution with a 90% activity retention with the membrane. The stability of the polyethylene-glycol-polylactic-acid bioactive membrane was found to be dependent on storage time at room temperature: activity of the membrane reduced to 79% approximately 2 months after fabrication. Future directions for this experiment include optimization of the electrospinning process to reduce needle-tip blockage from unspun polymer solution, or ‘icing’, and the reduction of the diffusion time for colorimetric glucose detection.