Purpose Colorectal cancer (CRC) is among the leading causes of cancer death worldwide. Graphene oxide (GO) plus doxorubicin (DOX) have low toxicity and facilitate drug carriage and provide enough surface. The GO-DOX anticancer effects against HCT-116 human CRC cells were compared with that of pure GO and DOX compounds. Methods Different concentrations of graphene oxide (GO), doxorubicin (DOX), and graphene oxide plus doxorubicin (GO-DOX) were prepared. The MTT test was conducted to determine the viability of cells and flow cytometry was performed following DOX, GO, and GO-DOX exposure. Expressions of caspase 3, Bax, and ATG5 autophagy-related genes were investigated using RT-qPCR technique. Results In the MTT test, DOX and GO at 100 mu g/mL and 40 mu g/mL exerted 50% cell death (LC50) against the HCT-116 cells. We observed significant differences in GO-DOX LC50 at concentrations of 1 (p = 0.003), 2.5 (p = 0.003), 5 (p = 0.00009), and 10 mu g/mL (p = 0.0001). The rate of apoptosis following GO, DOX, and GO-DOX included 24%, 31%, and 56%, respectively. The GO-DOX significantly increased the ATG5 (3.1-fold, p < 0.0001), caspase 3 (4.7-fold, p < 0.0001), and Bax (4.3-fold, p < 0.0001) gene expression. Conclusion The GO-DOX exerted anticancer effects against the HCT-116 cells via inducing the apoptosis and autophagy.