Yellow nose skate (Dipturus chilensis) cartilage extract was applied to upcycle and synthesize green gold nanoparticles (AuNPs) with spherical shapes (skAu). Collagen type II was identified in the extract by peptide analysis using LC-ESI-MS/MS. Doxorubine (DOX) was loaded onto skAu, and the average size of the DOX-loaded AuNPs (c-skAuD) was determined to be 14.6±0.5 nm by HR-TEM. For optimizing loading efficiency of DOX onto c-skAuD, reaction parameters such as reaction time and DOX concentration were evaluated. FT-IR spectra revealed a successful loading of DOX. Additionally, FT-IR characterization confirmed that chondroitin sulfate and collagen play a critical role in the synthesis of skAu. The cytotoxic effect of c-skAuD was evaluated on four different types of cancer cells (AGS, HeLa, A549 and MDA-MB-231). Among these cells, the cytotoxicity was the greatest on MDA-MB-231 cells. Interestingly, c-skAuD had the greatest cytotoxicity on MDA-MB-231 cells, followed by a positive control (citrate-reduced AuNPs, c-citAuD) and pristine DOX. Furthermore, the cytotoxicity was dependent on the incubation time and DOX concentration. ICP-MS was employed to determine cellular uptake of Au. When compared with AGS cells (15.1%), MDA-MB-231 cells showed approximately two-fold Au uptake (29.9%) of c-skAuD. However, the Au uptake of the positive control (c-citAuD) did not significantly differ between the MDA-MB-231 and AGS cells.