TiO2 has been generally studied for photocatalytic sterilization, but its antibacterial activities are limited. Herein, TiO2 nanospheres with rutile/anatase heterophase junctions are prepared by a wet chemical/annealing method. The large BET surface area and pore size are beneficial for the absorption of bacteria. The rutile/anatase heterojunctions narrow the bandgap, which enhances light absorption. The rutile/anatase heterojunctions also efficiently promote the photogenerated carriers’ separation, finally producing a high yield of radical oxygen species, such as •O2– and •OH, to sterilize bacteria. As a consequence, the obtained TiO2 nanospheres with rutile/anatase heterojunctions present an improved antibacterial performance against E. coli (98%) within 3 h of simulated solar light irradiation, exceeding that of TiO2 nanospheres without annealing (amorphous) and TiO2 nanospheres annealing at 350 and 550 °C (pure anatase). Furthermore, we design a photocatalytic antibacterial spray to protect the file paper. Our study reveals that the TiO2 nanospheres with rutile/anatase heterojunctions are a potential candidate for maintaining the durability of paper in the process of archival protection.