Bioconjugation methods offer very important tools in studying biological systems. Synthetic host–guest pairs provide an alternative and complementary conjugation method to bioorthogonal reactions and biological association pairs. Nevertheless, macrocyclic hosts that can be used for in situ capture are limited and often rely on extremely high binding affinities. Herein, we report an alternative bioorthogonal host–guest pair that relies on highly selective molecular recognition in water. The host, namely amide naphthotube, possesses a biomimetic cavity with inward-directing hydrogen bonding sites and shows selective and strong binding to the guest (2-phenyl pyrimidine) even in biological media. Through anchoring the tetraphenyl ethylene-modi fied hosts to cell surfaces, the bioorthogonal host–guest pair can be applied in cell surface recognition, cell–cell interactions, and tissue imaging in mice. The bioorthogonality is originated from the high binding selectivity of the biomimetic macrocyclic host, which is different from other known host–guest pairs that have been applied in biological systems. This research provides a new noncovalent bioconjugation tool and a new concept for designing bioorthogonal host–guest pairs for biological applications.