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Engineering cell surfaces with macromolecules offers the potential to manipulate and control their phenotype and function for cell-based therapies. In situconstruction and real-time evaluation of cell–macromolecule conjugates are vital for characterizing their dynamics, mobility, and function but remain a great challenge. Herein, we design a near-infrared (NIR) heptamethine cyanine (LS)-bearing dibenzocyclooctyne (DBCO) and norbornene (NB) in its structure for rapid and selective bioorthogonal “click” coupling to azide-labeled cells and tetrazine-functionalized macromolecular precursors. Specifically, only orthogonal dual “click” cell–macromolecule conjugates turn on NIR fluorescence, in which LS behaves as an AND logic gate, with azide- and tetrazine-derivatives being “input” and the emission intensity as the output. LS enables in situconstruction and real-time evaluation of the process and functional effects that macromolecules “graft to” cells with high cytocompatibility. This probe is tailor-made for live-cell microscopy technologies, which may open new opportunities for promoting further developments in cell-tracking and cell-based therapies.