Human　serum　albumin　(HSA)　plays　a　pivotal　role　in　various　physiological　processes　of　humans,　and　is　generally　considered　to　be　one　of　the　early　signs　of　many　diseases.　Although　dysfunction　of　intracellular　lysosome　is　accompanied　in　those　diseases,　the　regulation　mechanism　of　HSA　in　lysosomes　still　need　to　be　explored.　In　this　work,　we　report　a　novel　near-infrared　naphthalimide　probe　NI-1　that　specifically　detects　HSA　in　intracellular　lysosomes　with　the　＂turn-on＂　fluorescent　sensing　modes.　In　the　cell,　the　N-containing　malononitrile　group　of　NI-1　could　bind　to　the　lysosome.　Moreover,　NI-1　can　specifically　enter　the　site　II　hydrophobic　cavity　of　HSA　in　lysosome.　Due　to　the　binding　force　between　NI-1　and　HSA,　strong　steric　hindrance　and　the　hydrophobic　pocket　inside　HSA　inhibit　the　twisted　internal　charge　transfer　(TICT)　effect　in　the　probe　itself.　Therefore,　NI-1　emits　strong　red　fluorescence.　More　importantly,　NI-1　can　effectively　localize　bioimaging　of　exogenous　and　endogenous　HSA　in　lysosome.　In　addition,　the　novel　probe　NI-1　achieved　a　much　high　selectivity　for　HSA　over　bovine　serum　albumin　(BSA),　and　the　interaction　mechanism　between　probe　NI-1　and　HSA　or　BSA　site　II　was　explained　for　the　first　time　through　molecular　docking　methods.　These　results　indicate　that　the　probe　NI-1　has　great　potential　in　exploring　further　function　of　HSA　for　pharmacy　and　medicine.