Until now, the afterglow emissions of most developed near infrared (NIR)-emitting persistent luminescent nanoparticles (NPLNPs) were located at approximately 700 nm, at the edge of the first tissue transparency window (from 650 to 900 nm), which resulted in relatively low tissue penetration and signal-to-noise ratio (SNR) for in vivo imaging. Herein, 5 nm ZnSn₂O₄:Cr,Eu (ZSO) NPLNPs with NIR afterglow emission at 800 nm are synthesized via a direct aqueous-phase synthesis method. The longer NIR afterglow emission of ZSO NPLNPs can easily penetrate approximately 3 cm of pork tissue. Furthermore, even though the backbones blocked part of the NIR afterglow light, high SNR (25.5) in vivo images of the backs of mice can be observed and can be maintained for more than 15 min. The ZSO nanoprobes conjugated with folic acid exhibited excellent in vitro and in vivo tumor targeting capacity, which was advantageous for accurate tumor diagnosis. More importantly, the ZSO NPLNPs can be re-excited in situ and in vivo using NIR light to realize renewable near-infrared persistent luminescence in vivo, which was helpful for very long term and higher SNR in vitro and in vivo imaging.