Cancer is important cause of mortality, due to difficulty in finding safe, economical and specific effective therapeutic targets. The aim was to investigate the cytotoxicity of hybrid gold-chitosan nanospheres (Au@CsNs) on different cell lines using chitosan. Au@CsNs were analyzed by Zeta potential, UV-Visible spectroscopy, TEM and FTIR. Absorbance at 522 nm correlated to surface plasmon resonance (SPR) of AuNPs. TEM images show that Au@CsNs are spherical, with an average size of 12 ± 2 nm. Results of DPSS (diode pumped solid-state) laser irradiation show the photothermal laser stability of Au@CsNs. Au@CsNs showed anticancer effect using SRP assay on different cancer cell lines HCT, FADU, H460, and HEPG2. Our results suggest that Au@CsNs have an antiproliferative effect as there were a significant decrease in cell viability but this depends on the type of cancer cell line. In conclusion, Au@CsNs exhibited great anticancer activity subsequently it will be used in drug delivery and photothermal therapy in future work.