A two‐stage mediated near‐infrared (NIR) emissive supramolecular assembly for lysosome‐targeted cell imaging is presented. 4,4′‐Anthracene‐9,10‐diylbis(ethene‐2,1‐diyl))bis(1‐ethylpyridin‐1‐ium) bromide (ENDT) was synthesized as an organic dye with weak fluorescence emission at 625 nm. When ENDT complexes with cucurbit8uril (CB8), this binary supramolecular complex assembles into nanorods with a near‐infrared fluorescence emission (655 nm) and fluorescence enhancement as the first stage. Such supramolecular complexes interact with lower‐rim dodecyl‐modified sulfonatocalix4arene (SC4AD) to form nanoparticles for further fluorescence enhancement as the second stage. Furthermore, based on a co‐staining experiment with LysoTracker Blue, such nanoparticles can be applied in NIR lysosome‐targeted cell imaging. Supramolecular assemblies were designed with initially weak 625 nm near‐IR emission that was enhanced and shifted to 655 nm in two stages by cucurbit8uril (CB8) and amphiphilic calixarene SC4AD. This system was used for lysosome‐targeted cell imaging.