Na-O2 batteries are highly valued batteries with high energy density and low over-potentials, but limited by the threat of sodium dendrites. In this paper, a new trilayer separator with TiO2 nanoparticles as the middle layer is proposed, which can effectively inhibit the growth of sodium dendrites. The TiO2 sandwiched separator was used in a Na-O2 battery, and increased its cycle life. This work provides a new approach to extend the cycle life of sodium-based rechargeable batteries with a sandwiched separator.