Au–Ft, as a green synthesized nanoparticle, is composed of a ferritin nanocage enclosing a pair of Au nanoclusters inside. Our previous study has demonstrated that Au–Ft can be an excellent fluorescent probe for whole body imaging of mice with kidney specific targeting. But, the accurate localization of Au–Ft in kidney is still absent. In the current study, we detected and assessed the cellular and subcellular localization of Au–Ft in renal cortex and medulla of nu/nu mice after tail vein injection by using Nuance optical system (CRi, Woburn, USA) and inForm intelligent image analysis software based on single cell segmentation. We obtained the fluorescence intensity and cellular location of kidney-targeting Au–Ft probe in particular cell of renal glomerulus or renal tubules, which provided valuable proofs to clarify the mechanism of Au–Ft selective enrichment in kidney and the associated metabolic processes.