Here, we report the novel use of the ethonolic leaf extract of Piper betle for gold nanoparticle (AuNP) synthesis. The successful formation of AuNPs was confirmed by UV-visible spectroscopy, and different parameters such as leaf extract concentration (2%), gold salt concentration (0.5 mM), and time (18 s) were optimized. The synthesized AuNPs were characterized with different biophysical techniques such as transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and energy-dispersive X-ray spectroscopy (EDX). TEM experiments showed that nanoparticles were of various shapes and sizes ranging from 10 to 35 nm. FT-IR spectroscopy revealed that AuNPs were functionalized with biomolecules that have primary amine group –NH2, carbonyl group, –OH groups, and other stabilizing functional groups. EDX showed the presence of the elements on the surface of the AuNPs. FT-IR and EDX together confirmed the presence of biomolecules bounded on the AuNPs. Cytotoxicity of the AuNPs was tested on HeLa and MCF-7 cancer cell lines, and they were found to be nontoxic, indicating their biocompatibility. Thus, synthesized AuNPs have potential for use in various biomedical applications.