Based on the AuNCs hydrophobic paper substrate and enzymatic cleavage strategy, a rapid, highly sensitive and specific method for detecting gastric cancer (GC)-associated miRNA markers was developed. First, gold nanocages (AuNCs) were assembled on the hydrophobic release paper surface to form a Array style AuNCs substrate with rich "hotspots". Then, miR-21 complementary single-stranded nucleotide ssDNA-21 is modified on the surface of the arrayed AuNCs substrate, followed by ligation of the 4-MBA-labled Au - Ag nano-shuttles (Au-AgNSs) to form a complex structure of the Au-AgNSs@4-MBA@ssDNA-21@AuNCs substrate. Finally, after hybridization between ssDNA-21 and miR-21, DNA phosphodiester bonds in ssDNA-21 miR-21 double stranded DNA were selectively cleaved using double stranded specific cleavage enzyme (DSN) for SERS detection. The linear range of the SERS microfluidic chip for detecting miR-21 is 1 fM~1 nM, with a detection limit (LOD) of 0.34 fM. In addition, the SERS sensor is easy to operate, with a testing process of only 30 minutes, and exhibits good specificity and reproducibility. By using this SERS sensor, we successfully detected and distinguished the difference of miR-21 expression levels in serum of healthy people and GC patients. The accuracy of SERS was validated by real-time quantitative polymerase chain reaction (qRT PCR).