Intratumoral (IT) injection of chemotherapeutics into needle-accessible solid tumors as breast cancer and head and neck squamous cell carcinomas (HNSCC) can directly localize the anticancer drug in the tumor site, thus increasing its local bioavailability and reducing its undesirable effects compared to systemic administration. In this study, graphene oxide (GO)-based chitosan/β-glycerophosphate (CS/GP) thermosensitive injectable composite hydrogels (CH) were prepared and optimized for localized controlled delivery of doxorubicin (DOX). A quality-by-design (QbD) approach was used to study individual and combined effects of several formulation variables to produce optimal DOX-loaded CH (DOX/opt CH) formulation with predetermined characteristics, including gelation time, force required for injection (injectability), degree of porosity, and swelling capacity. The optimization of CHs was carried out using Design-Expert® software (Version 13.0, Stat-Ease Inc., Minneapolis, MN, USA). The design involved investigating the effect of the concentration of GO (0–0.1% w/v X1), the concentration of CS (1.5–2% w/v X2), and the CS:GP ratio (2:1 and 3:1 v/v X3) as CMAs for the selected CQAs. Planning the levels of the independent variables and analysis of the output data with the minimal number of experimental runs was carried out using a D-optimal response surface design. The hydrogel formulations were optimized for the measured responses: gelation time (Y1), injectability (Y2), degree of porosity (Y3), and swelling capacity (Y4) with the target response set at a gelation time of around 3 min, minimum injection force, the lowest degree of porosity and maximum swelling percentage. The surface morphology of DOX/opt CH, chemical interaction between its ingredients and in vitro release of DOX in comparison to GO-free CS/GP CH were investigated. Cell viability and cellular uptake after treatment with DOX/opt CH were studied on human breast cancer cell lines, MCF 7 and MDB-MB-231 and a human epithelial cel