Objective The fluorescent dye Nile red (NR) is commonly used in quantitative analysis of lipids in bacteria, fungi, and microalgae. In this study, NR was encapsulated in a nanoemulsion to investigate the tumor targeting and tissue distribution of the nanoemulsion in BALB/ c nude mice bearing H1688 tumors. Methods To evaluate the prospects of NR as a bioimaging marker for nanoemulsions, its cytotoxicity was investigated in H1688 cells with a CCK-8 assay. An NR suspension (NRS) and NR nanoemulsion NRNE(O) were given by gavage. The dynamic fluorescence intensity distribution of NR in mice was observed with an in vivo fluorescence imaging system. Results The H1688 cells exhibited over 85% viability after incubation with a range of NRNE(O) concentrations for 24 h. The results demonstrated that NRNE(O) absorption in mice and the amount accumulated in tumor tissue were both higher than in the NRS group.Conclusions NRNE(O) has a low cytotoxicity and good biocompatibility. NR in a nanoemulsion combined with an in vivo fluorescence imaging system can reveal the dynamic distribution of the nanoemulsion in mice vividly and reliably in real time. The results of this study indicate that NR has significant application potential as a tracer for the bioimaging of nanoparticles in vivo.