SCOBY disc is a unique structure of bacteria and fungi. It was known in the mid-Seventies in Egypt as “Tea fish." To the author's knowledge, no reports since then were published to use them in producing nanoparticles worldwide. The addition of 1mM of (HAuCl₄.3H₂O) solution into the cell-free extract (CFE) of SCOBY disc grew on black tea medium, resulted in gold nanoparticles AuNPs at 25ºC (Au³⁺→Au⁰) momentarily. The surface plasmon resonance peaks of the gold colloidal solution produced showed an absorption peak at 530 nm. (TEM) exhibited AuNPs in spherical and triangle shapes (12 to 35 nm). X-ray diffraction reflected four distinct peaks at 2θ = 38.1°, 44.3°, 64.5°, and 77.7°. All four peaks corresponded to standard Bragg reflections (111), (200), (220), and (311) of face center cubic. (FTIR) analysis showed the presence of two amide groups in 1721 & 1033 respectively, which appeared to be responsible for the stability of gold nanoparticles. The mean particle size and polydispersity index (PDI) of AuNPs induced by SCOBY were 61.23 ± 0.416 with a negative charge -10.2 ± 1.73 mV that cause electrostatic repulsion among the nanoparticles and good colloidal stability. Moreover, the effect of AuNPs on inducing apoptosis in liver cancer cells HepG2. 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay showed that AuNPs produced had selective toxic effects to HepG2 cells with IC-50 value 176 µM/mL compared to control cells. These results suggested the use of those green synthesized AuNPs in cancer treatment and further studies are recommended.