Simultaneous detection of multiple trace cancer associated serum miRNA biomarkers is considered as a feasible method for early cancer screening and diagnosis. In the present work, an ultrasensitive SERS sensor was prepared based on an Ag nanorod array SERS substrate by assembling special hairpin-shaped molecular beacons (MBs) for the detection of multiple lung cancer-related miRNA biomarkers. The portable SERS sensor exhibits excellent performance for the qualitative and quantitative detection of miRNAs, with advantages of ultra-sensitivity, good specificity, uniformity, reproducibility and stability, as well as remarkable reusability. By monitoring the SERS signal quenching of the MBs in the presence of target miRNA biomarkers, three lung cancer related-miRNAs (miRNA-21, miRNA-486, and miRNA-375) in buffer and human serum were simultaneously assayed using the SERS sensor array, and the limits of detection of the three miRNAs in human serum are 393 aM, 176 aM, and 144 aM, respectively. The reliable results demonstrate that the proposed SERS sensor array can be a promising candidate with great potential for the screening and clinical diagnosis of cancer in the early stage.