Host–guest complexation between calix5arene and aggregation‐induced emission luminogen (AIEgen) can significantly turn off both the energy dissipation pathways of intersystem crossing and thermal deactivation, enabling the absorbed excitation energy to mostly focus on fluorescence emission. The co‐assembly of calix5arene amphiphiles and AIEgens affords highly emissive supramolecular AIE nanodots thanks to their interaction severely restricting the intramolecular motion of AIEgens, which also show negligible generation of cytotoxic reactive oxygen species. In vivo studies with a peritoneal carcinomatosis‐bearing mouse model indicate that such supramolecular AIE dots have rather low in vivo side toxicity and can serve as a superior fluorescent bioprobe for ultrasensitive fluorescence image‐guided cancer surgery. Calix5arene‐based supramolecular AIE nanodots were synthesized with high quantum yields in water by virtue of the host–guest complexation. The absorbed excitation energy was mostly focused on fluorescence emission, leading to an ultrahigh signal‐to‐background ratio in fluorescence‐image‐guided cancer surgery.