Near infrared (NIR)-emitting persistent luminescent nanoparticles (NPLNPs) have attracted much attention in bioimaging because they can provide long-term in vivo imaging with a high signal-to-noise ratio (SNR). However, conventional NPLNPs with large particle sizes that lack modifiable surface groups suffer from many serious limitations in bioimaging. Herein, we report a one-step synthesis of amino-functionalized ZnGa2O4:Cr,Eu nanoparticles (ZGO) that have an ultrasmall size, where ethylenediamine served as the reactant to fabricate the ZGO as well as the surfactant ligand to control the nanocrystal size and form surface amino groups. The ZGO exhibited a narrow particle size distribution, a bright NIR emission and a long afterglow luminescence. In addition, due to the excellent conjugation ability of the surface amino groups, the ZGO can be easily conjugated with many bio-functional molecules, which has been successfully utilized to realize in vitro and in vivo imaging. More importantly, the ZGO achieved re-excitation imaging using 650 nm and 808 nm NIR light in situ, which is advantageous for long-term and higher SNR bioimaging.