Titanium dioxide nanoparticles (TiO2NPs) were produced using a crud flavonoid luteolin (LT) by the green process. Physicochemical characterizations of LT mediated synthesis of TiO2NPs (LT-TiO2NPs) were analyzed by different techniques. The biocompatibility of LT-TiO2NPs was evaluated by assessing their cytotoxicity, inhibition of protein denaturation, hemolysis effects, and membrane stabilization activity. The synthesized LT-TiO2NPs exhibited mostly rod-shaped morphology, rutile crystalline structure with length about 33.3–135 nm and width in 16.6–58 nm. Under LT-TiO2NPs treatments, the cell viability of cancer cells was decreased in a dose-dependent manner. According to the results, the IC50 values of LT-TiO2NPs were between 12.5–25 lg/mL for A375 and MCF-7 cancer cells, and its value was below 50 lg/mL for skin fibroblast. These NPs also induced intracellular reactive oxygen species (ROS) generation and caused mitochondrial disruption in cancer cells, leading to the induction of intrinsic apoptosis. These biosynthesized NPs did not show denaturation of bovine serum albumin (BSA) protein and hemolytic or heatinduced hemolytic effects, which confirmed their membrane stabilization activity without damaging red blood cells (RBCs) membrane integrity. Therefore, this research provides valuable results in the case of green synthesis of LT-TiO2NPs and their specific properties, biocompatibility, and their potential to be used in biomedical fields.