Cell imaging with fluorescent, magnetic or bimodal nanoparticles is currently undergoing a rapid development. The goal of this study was to evaluate the feasibility and efficiency of cell imaging by using a novel kind of organic fluorescent nanorods without any other dye staining or antibody labeling. Perylene-3, 4, 9, 10-tetracarboxdiimide (PDI) nanorods with uniform diameters (ca. 50 nm) and in a length range of 100–600 nm were fabricated by a template-assisted self-assembly method. The PDI nanorods showed a strong fluorescent emission in the visible range and favorable dispersity in aqueous solution. Human hepatoma (HepG2) cells, Hela cells and human embryonic fibroblasts (HEFs) were chosen as models to investigate the cytotoxicity and the cell imaging effect. MTT assays demonstrated the cytotoxicity of PDI nanorods was concentration-dependent and the 72 h IC50 value were in the range of 42–55 μg/mL which is much higher than that of reported quantum dots (QDs). Particularly, con-focal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) observations indicated PDI nanorods were able to be internalized into the cells very readily and mostly located in the cytoplasm. Moreover, they were found exhibiting a stable, bright fluorescence as long as several hours within the cells. Besides, the uptake of PDI nanorods was found through an energy-dependent, endocytosis-mediated process.