Activatable　near-infrared　(NIR)　fluorescent　probes　possess　advantages　of　high　selectivity,　sensitivity,　and　deep　imaging　depth,　holding　great　potential　in　the　early　diagnosis　and　prognosis　assessment　of　tumors.　However,　small-molecule　fluorescent　probes　are　largely　limited　due　to　the　rapid　diffusion　and　metabolic　clearance　of　activated　fluorophores　in　vivo.　Herein,　we　propose　an　efficient　and　reproducible　novel　strategy　to　construct　activatable　fluorescent　nanoprobes　through　bioorthogonal　reactions　and　the　strong　gold-sulfur　(Au-S)　interactions　to　achieve　an　enhanced　permeability　and　retention　(EPR)　effect,　thereby　achieving　prolonged　and　high-contrast　tumor　imaging　in　vivo.　To　demonstrate　the　merits　of　this　strategy,　we　prepared　an　activatable　nanoprobe,　hCy-ALP@AuNP,　for　imaging　alkaline　phosphatase　(ALP)　activity　in　vivo,　whose　nanoscale　properties　facilitate　accumulation　and　long-term　retention　in　tumor　lesions.　Tumor-overexpressed　ALP　significantly　increased　the　fluorescence　signal　of　hCy-ALP@AuNP　in　the　NIR　region.　More　importantly,　compared　with　the　small-molecule　probe　hCy-ALP-N3,　the　nanoprobe　hCy-ALP@AuNP　significantly　improved　the　distribution　and　retention　time　in　the　tumor,　thus　improving　the　imaging　window　and　accuracy.　Therefore,　this　nanoprobe　platform　has　great　potential　in　the　efficient　construction　of　biomarker-responsive　fluorescent　nanoprobes　to　realize　precise　tumor　diagnosis　in　vivo.　©　2024　American　Chemical　Society.