We describe the fabrication and characterization of poly(ethylene glycol) (PEG) hydrogel spheres containing the enzyme horseradish peroxidase (HRP) for application as optical nanosensors for hydrogen peroxide. HRP was encapsulated in PEG hydrogel spheres by reverse emulsion photopolymerization, yielding spheres with a size range from 250 to 350 nm. Encapsulated HRP activity and sensitivity to hydrogen peroxide were investigated by the Amplex Red assay based on the fluorescence response as a function of H2O2. These HRP-loaded spheres were then introduced to murine macrophages with Amplex Red in the culture media. After phagocytosis, the biocompatibility of spheres was determined by live cell staining using calcein AM (5 microM). The HRP-loaded PEG hydrogel spheres were activated (i.e., fluorescent oxidized Amplex Red produced within the spheres) by oxidative stresses such as exogenous H2O2 (100 microM) and lipopolysaccharide (1 microg/mL), which induced the production of endogenous peroxide inside macrophages. The results presented here indicate that after polymerization, the enzyme activity of HRP was still maintained and that using these HRP-containing nanospheres, peroxide production could be sensed locally within cells.