The premise that long afterglow can be applied is its duration, and the persistent duration is closely related to the depth of the traps. Therefore, the stable deep traps are the key to obtain long persistent luminescence. Based on this, a strategy that X-ray excites high-gap phosphors to achieve long persistent luminescence is firstly proposed. Herein, rare earth (RE) ions doped YPO4 phosphor is adopted as the research object as RE ions can form stable and deeper defect centers or luminescent centers in high bandgap materials. Furthermore, the efficient method of enhancing persistent luminescence is designed so that introducing Tb3+ ions into YPO4:Sm3+ crystals forms tightly bound excitons, which modulates the depth of defect centers (Sm3+ ions), improving the afterglow behavior from Sm3+ ions for more than two days, which is approximately 14 times stronger than the afterglow of YPO4:Sm3+ phosphors itself. Finally, highly efficient in vivo deep tissue bioimaging was successfully achieved through mouse tail intravenous injection. The results indicate that the YPO4:Sm3+,Tb3+ phosphor possesses great promise in the field of in vivo imaging.