Titanium dioxide (TiO2) nanosheets had received attentions in photodynamic therapy due to unique electronic structure and high surface activity. However, it is still not clear about the biological response and toxicity of two dimensional (2D) nanomaterials. Herein, in vivo toxicity of TiO2 nanosheets, such as biodistribution, hematology, biochemistry and pathology, were evaluated at the dose of 10 mg/kg by intraperitoneal injection up to 30 days. It was found that TiO2 could gradually accumulate in liver and spleen with increase of exposure time, which is due to large size induced absorption from reticuloendothelial system (RES). Furthermore, the hematological data indicated that no significant difference was found. However, the biochemistry showed that liver indicator, AST, presented significant difference after 30 days compared with control mice. The present work revealed that 2D TiO2 nanosheets did not cause the appreciable toxicity, but induced the accumulation in the liver and slight abnormality of liver with increasing exposure time.