Fluorescent　probes　are　essential　for　optical　imaging　and　have　been　extensively　employed　for　precise　cancer　diagnosis　studies.　β-galactosidase　(β-gal)　serves　as　a　primary　biomarker　for　ovarian　cancer　and　has　been　utilized　to　develop　imaging　probes　for　accurate　tumor　diagnosis.　However,　traditional　small　molecular　probes　have　limitations　in　terms　of　rapid　diffusion　and　metabolic　clearance　from　the　target　lesion,　resulting　in　a　short　imaging　window　and　compromised　tumor-to-background　ratios　(TBR).　Herein,　we　integrated　an　enzyme-instructed　in　situ　self-assembly　strategy　to　construct　Gal-IRFF,　a　small　molecule-based　activatable　near-infrared　(NIR)　fluorogenic　probe.　Upon　cleavage　by　endogenous　β-gal　overexpressed　in　ovarian　cancer　cells,　IRFF　exhibited　enhanced　NIR　fluorescence　signals　and　self-assembled　into　nanoparticles　through　intermolecular　interactions　of　the　Phe-Phe　(FF)　dipeptide　moiety,　which　facilitated　probe　accumulation　and　retention　within　the　tumor　lesion.　Compared　with　the　small　molecule　probe　Gal-IR,　our　proposed　self-assembly　probe　Gal-IRFF　demonstrated　a　lower　limit　of　detection　(LOD)　towards　β-gal　and　showed　remarkable　improvements　in　distribution　and　retention　time　within　SKOV3　cells　in　vitro　and　tumors　in　vivo,　thereby　providing　a　long-term　imaging　window　for　real-time　monitoring　β-gal　levels　in　ovarian　tumors.　Therefore,　this　study　highlights　the　potential　of　an　enzyme-instructed　self-assembly　fluorogenic　probe　design　approach　for　achieving　precise　tumor　diagnosis　in　vivo.　©　2024　Elsevier　B.V.