Zn₂Ga_3−x−yCr_xNd_yGe_0.75O₈ (ZGGO:Cr_x,Nd_y) persistent luminescent nanoparticles (PLNPs) (x = 0, 0.02; and y = 0, 0.01, 0.02, 0.03, 0.04) were synthesized via a hydrothermal method in combination with a following heat treatment under vacuum. The effects of Nd³⁺ concentration on chemical composition, size distribution, luminescence property, the persistent energy transfer from Cr³⁺ to Nd³⁺ and afterglow imaging were studied in detail. When the Nd³⁺ concentration was increased from 0 to 0.04, the particle size increased from 45.4 to 86.2 nm. The afterglow emissions in the NIR-I (696 nm) and NIR-II (1067 nm) regions, which are ascribed to the ²E, ⁴T₂ → ⁴A₂ transitions of Cr³⁺ and the ⁴F_3/2 → ⁴I_11/2 transition of Nd³⁺, respectively, were simultaneously acquired after stopping the 635 nm excitation. Among the nanoparticles with different concentrations, ZGGO:Cr_0.02,Nd_0.02 exhibits the strongest NIR-II afterglow intensity and the corresponding energy transfer efficiency from Cr³⁺ to Nd³⁺ is found to be 25.9%. In addition, enhanced X-ray excited afterglow imaging can be observed in Nd³⁺/Cr³⁺ codoped nanoparticles dispersed in water, human serum albumin solution and simulated lysosomal environment compared to the Cr³⁺ singly doped nanoparticles. Renewable NIR afterglow imaging was realized through an X-ray reexcitation strategy. In particular, both the X-ray excitation strategy accompanied by NIR-I afterglow emission and the red light (635 nm) excitation strategy accompanied by NIR-II afterglow emission exhibit high tissue penetration capability. This study provides a further understanding of how to develop a suitable strategy for realizing deep tissue autofluorescence-free bioimaging.