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To sensitively detect multiple and cross-species disease-related targets from a single biological sample in a quick and reliable manner is of high importance in accurately diagnosing and monitoring diseases. Herein, a surface-enhanced Raman scattering (SERS) sensor based on a functionalized multiple-armed tetrahedral DNA nanostructure (FMTDN) immobilized silver nanorod (AgNR) array substrate and Au nanoparticle (AuNP) SERS tags is constructed to achieve both multiplex detection and enhanced sensitivity using a sandwich strategy. The sensor can achieve single, dual, and triple biomarker detections of three lung cancer-related nucleic acid and protein biomarkers, i.e. , miRNA-21, miRNA-486 and carcinoembryonic antigen (CEA) in human serum. The enhanced SERS signals in multiplex detections are due to the DNA self-assembled AuNP clusters on the silver nanorod array during the assay, and the experimentally obtained relative enhancement factor ratios, 150 for AuNP dimers and 840 for AuNP trimers, qualitatively agree with the numerically calculated local electric field enhancements. The proposed FMTDN-functionalized AgNR SERS sensor is capable of multiplex and cross-species detection of nucleic acid and protein biomarkers with improved sensitivity, which has great potential for the screening and clinical diagnosis of cancer in the early stage.