Glutathione (GSH) capped Iron-doped zinc oxide nanoparticles (GSH-Fe: ZnO NPs)at varying GSH concentrations (0.002, 0.004, 0.006, 0.008, 0.01, 0.012, 0.014M) were successfully synthesized through sol-gel route to study their effects on the structural, optical,and morphological properties. Characterization was carried out using X-ray diffractometer (XRD), photoluminescence (PL) spectroscopy, and Ultraviolet-Visible spectrometer (UV- Vis). While Fe: ZnO NPs have interesting optical and photoluminescence properties, their usage in bio-imaging are still limited by surface imperfections and poor biocompatibility. More surface functionalization approaches, such as biomolecule-based capping, are needed to improve their performance and biocompatibility.XRD confirmed the retention hexagonal wurtzite structure and successful incorporationof Fe into the ZnO lattice. Optical analysis showed systematic absorbance modulation, a blue shift, and reduced optical bandgap (3.0 eV) at 0.008 M GSH capping. The PL analysis revealed that GSH capping enhances emission intensitybecause GSH reduces non-radiative recombination and facilitates light emission. The PL spectra deconvolution reveals the presence of near-band emissions (NBE) and deep-level emissions (DLE). FTIR analysis revealed that glutathione capping to the Fe-doped ZnO NPs occurred through N–H, C=O, and COO⁻ bonds. This result not only addresses the existing knowledge gap on surface functionalization but also establishes a foundation for future nanomedical applications of these NPs.