Abstract The rigidity of traditional solid-state surface-enhanced Raman spectroscopy (SERS) substrate hampers their application in the curved structure for nonplanar surface test and in-situ detection. Traditionally, the flexible Raman substrates are often prepared by transferring printing of patterned nanoparticles on the flexible materials such as polymer, paper, etc. However, the replicate patterns are often produced by high-cost instruments. In this study, a low-cost and flexible SERS substrate is prepared by using a microcontact printing technology to transfer three-phase-assembled nanoparticles on a polydimethylsiloxane film, which can stabilize the assembled nanoparticles. Combining with the endonuclease Nt.BbvCI assisted amplification method, a SERS biosensor is constructed for microRNA 21 (miRNA 21) assay. This platform presents a wide dynamic range (100 fM ∼1 nM), achieving a fabulous sensitivity with limit of detection of 11.96 fM for miRNA 21. Furthermore, after being bent 90° for 50 times, the Raman intensity of the flexible substrate shows a negligible change. This versatile flexible substrate exhibits considerable potential for SERS analysis, which also opens a new avenue for preparing flexible devices.