Herein, we fabricate the graphene oxide-supported nanofilms coated by roughened nanoboxes (GO@AuAgRNB) for the ultrasensitive and simultaneous determination of multiple stroke subtype-specific biomarkers. Initially, Au-Ag roughened nanobox (AuAgRNB) with abundant coupling and tip hotspots is prepared by the partial surface passivation strategy. AuAgRNB is uniformly, densely and firmly assembled onto graphene oxide (GO) by metal-sulfur bonds, generating extensive high-density hotspots. Owing to electromagnetic and chemical enhancement, the surface enhanced Raman scattering (SERS) activity of GO@AuAgRNB is greatly improved with the enhancement factor of 5.78 × 10