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It is here engineered a portable transdermal biosensor comprising antibody\u2010functionalized hierarchical porous microneedles (hp\u2010MNs) that enables on \u2010needle quantitatively detect human epididymis protein 4 (HE4) and cancer antigen 125 (CA125) via fluorescence immunoassay. Fabricated through a novel phase separation micro\u2010molding technique, the biomimetic microneedles feature a coral\u2010like hierarchical inter\u2010connective porous structure with noteworthy liquid absorption capacity and high antibody\u2010binding density, enabling reliable in vivo biomarkers enrichment and detection with nanomolar sensitivity to differentiate disease from benign biofluids. Furthermore, a groove structure allows concurrently detection of multiple tumor biomarkers at identical regions without cross\u2010influence in a single application, enhancing sensitivity and precision. 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