MicroRNAs are widely used as tumor markers for cancer diagnosis and prognosis. Herein, a multiple signal amplification sandwich-type SERS biosensor for femtomolar detection of miRNA is reported. The signal unit consisted of giant Au vesicles, DNA sequences and deposited silver nanoparticles. The giant Au vesicles provided large-volume hot spots because of sharp tips and abundant hotspot gaps, thus enhancing the electromagnetic intensity for the SERS performance. Further silver stain would easily lead to second-stage amplification of Raman signal. In addition, more SERS signal molecules R6G adsorbed on the signal unit with the aid of HCR and the controlled nanogaps between adjacent AgNPs, brought about the third-stage amplification. The capture unit, prepared by immobilizing the capture probe (CP) on the FeO@AuNPs, could easily capture target miRNA and greatly simplify the separation step to improve reproducibility. The higher concentration of target miRNA definitely formed more sandwich-type structures with combination of capture unit and signal unit, resulting in multiple amplification of SERS signals. The proposed multiple signal amplification sandwich-type SERS biosensor could detect miRNA-141 at the femtomolar level with a low detection limit of 0.03 fM. Meanwhile, it exhibited high selectivity and accuracy, even for practical analysis in human serum. Therefore, the designed multiple signal amplification sandwich-type SERS biosensor would be a very promising alternative tool for the detection of miRNA and analogs in the field of biomedical diagnosis.