Reproducible surface enhanced Raman scattering (SERS) enhanced performance, affected by molecular orientations in plasmonic nanostructures, attracts more attention than SERS detection sensitivity for satisfying requirements of practical applications. In this work, by coating silver films on the inverted cone-shaped nanopore array anodic aluminum oxide membranes, we provide a way to study the effect of molecular orientations on SERS enhancement with mapping technology. SERS enhanced performance of the fabricated substrates is assessed with trace Rhodamine 6G detection, enhancement factor calculation and 3D finite-difference time-domain simulation respectively. It is found that inverted cone-shaped nanopore and silver films both contribute to the highly SERS enhancement. And special inverted cone-shaped nanopore plasmonic structures limit molecular vibrations so as to affect Raman scattering enhancement. These findings can help us to understand the electromagnetic mechanism of molecular orientations on SERS enhancement, so as to design highly reproducible SERS substrates for various applications.