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introduction: The application of gold nanoparticles (GNPs) in medicine is expanding as an effective therapeutic and diagnostic compound. Different polysaccharides with high biocompatibility and hydrophilic properties have been used for synthesis and capping of GNPs. Chondroitin sulfate (CHS) as a polysaccharide possesses a wide range of biological functions e.g. anti-oxidant, anti-inflammation, anti-coagulation, anti-atherosclerosis, anti-thrombosis with insignificant immunogenicity and has not been used for the green synthesis of GNPs. Methods: GNPs were synthesized using CHS, and their physicochemical properties were evaluated. The antibacterial activity of CHS-GNPs was estimated against both gram-positive and gram-negative bacteria. The cytotoxicity of CHS and CHS-GNPs was obtained by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) test, and the electrocatalytic activity of CHS-GNPs was investigated. The blood compatibility was evaluated by the in vitro hemolysis assay. Results: The absorption band at 527 nm reveals the reduction of Au3+ into GNPs. The transmission electron microscopy (TEM) image displays the spherical shape of GNPs in the range of 5.8–31.4 nm. The CHS and CHS-GNPs at 300 µg/mL revealed a maximum DPPH (1, 1-diphenyl-2-picrylhydrazyl) scavenging activity of 73% and 65%, respectively. CHS-GNPs showed antibacterial activity against Bacillus subtilis, while CHS has no antibacterial activity. CHS-GNPs exhibited a cytotoxicity effect against MDA-MB-468 and βTC3 cancer cell lines, and the electrochemical study indicated a significant increase in electrocatalytic properties of CHS-GNPs coated electrode compared by the bare electrode. The hemolysis test proved the blood compatibility of CHS-GNPs. Conclusion: The results indicate the advantages of using CHS to produce blood-compatible GNPs with antioxidant, cytotoxic, and electrochemical properties.