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In the present study, a green surface modification of gold nanoparticles (GNPs) using chondroitin sulfate (CHS) and chitosan (CS) to deliver an extended-release of doxorubicin (DOX) was proposed. Following synthesis of each step of unconjugated counterpart, including CHS-GNPs, DOX–CHS–GNP, and conjugated construct DOX–CHS–GNP-CS, physicochemical properties of the nanoparticles (NPs) were characterized by FT-IR, DLS, and TEM analyses, and the release of DOX was determined by using UV–Vis spectrometry. Then, NPs were effectively taken up by MDA-MB-468, βTC-3, and human fibroblast (HFb) cell lines with high release percent and without significant cytotoxicity. The DOX–CHS–GNPs and DOX–CHS–GNP-CS NPs showed a mean size of 175.8 ± 1.94 and 208.9 ± 2.08 nm; furthermore, a zeta potential of − 34 ± 5.6 and − 25.7 ± 5.9 mV, respectively. The highest release of DOX was 73.37% after 45 h, while in the absence of CS, the release of DOX was 76.05% for 24 h. Compared to CHS-GNPs, the presence of CS decreased the rate of sustained release of DOX and improved the drug release efficiency. The results demonstrated an excellent release and negligible cytotoxicity at high concentrations of CHS-GNP-CS. Consequently, in ovo assessment corroborated the efficacy of the green fabricated NPs proposed effective targeted delivery of DOX for anti-tumor therapy in vitro. Graphical Abstract