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.

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