As one of the most common chemical materials, titanium dioxide (TiO2) has been prepared and widely used for many years. Among all the applications, the biomedical applications of TiO2 have motivated strong interest and intensive experimental and theoretical studies, owing to its unique photocatalytic properties, excellent biocompatibility, high chemical stability, and low toxicity. Advances in nanoscale science suggest that some of the current problems of life science could be resolved or greatly improved through applying TiO2. This paper presents a critical review of recent advances in the biomedical applications of TiO2, which includes the photodynamic therapy for cancer treatment, drug delivery systems, cell imaging, biosensors for biological assay, and genetic engineering. The characterizations and applications of TiO2 nanoparticles, as well as nanocomposites and nanosystems of TiO2, which have been prepared by different modifications to improve the function of TiO2, are also offered in this review. Additionally, some perspectives on the challenges and new directions for future research in this emerging frontier are discussed.