This study reports the green synthesis, characterization, and cytotoxic evaluation of zinc oxide (ZnO) nanoparticles. ZnO nanoparticles have been synthesized using 3 mL of waste peel extracted from Allium cepa (onion) and designated as O3. Comprehensive characterization has been performed using XRD, UV–Visible, FTIR, FESEM, HRTEM, DLS, and fluorescence spectroscopy. The UV–Visible spectrum exhibited an absorption peak at 345 nm, confirming ZnO nanoparticle formation. XRD analysis shows a wurtzit