Wilson＇s　disease　(WD)　is　a　rare　inherited　disorder　of　copper　metabolism　with　pathological　copper　hyperaccumulation　in　some　vital　organs.　However,　the　clinical　diagnosis　technique　of　WD　is　complicated,　aggressive,　and　time-consuming.　In　this　work,　a　novel　ratiometric　photoacoustic　(PA)　imaging　nanoprobe　in　the　NIR-II　window　is　developed　to　achieve　noninvasive,　rapid,　and　accurate　Cu2+　quantitative　detection　in　vitro　and　in　vivo.　The　nanoprobe　consists　of　Cu2+-responsive　IR970　dye　and　a　nonresponsive　palladium-coated　gold　nanorod　(AuNR-Pd),　achieving　a　concentration-dependent　ratiometric　PA970/PA1260　signal　change.　The　urinary　Cu2+　content　is　detectable　within　minutes　down　to　a　detection　limit　of　76 ×　10−9　m.　This　report　acquisition　time　is　several　orders　of　magnitude　shorter　than　those　of　existing　detection　approaches　requiring　complex　procedure.　Moreover,　utilizing　the　ratiometric　PA　nanoprobe,　PA　imaging　enables　biopsy-free　measurement　of　the　liver　Cu2+　content　and　visualization　of　the　liver　Cu2+　biodistribution　of　WD　patient,　which　avoid　the　body　injury　during　the　clinical　Cu2+　test　using　liver　biopsy　method.　The　NIR-II　ratiometric　PA　detection　method　is　simple　and　noninvasive　with　super　precision,　celerity,　and　simplification,　which　holds　great　promise　as　an　alternative　to　liver　biopsy　for　clinical　diagnosis　of　WD.　©　2021　Wiley-VCH　GmbH