DNA and amino acids are important biomolecules in living organisms. Probing such biomolecules with structural characters can provide valuable information for life study. Here, gold plasmonic nanopores (GPNs) with high SERS activity (a local enhancement factor higher than 109) are synthesized at the tip of a glass nanopipette. An electric field drives individual molecules to translocate through the GPNs, which enables in situ collection of the surface-enhanced Raman scattering (SERS). Nonresonant biomolecules, including nucleobases, amino acids, and oligonucleotides (DNA), with single nucleobase differences can be distinguished. The intensity of SERS is tunable by modulating the affinity between DNA and the GPNs. The present study shows the feasibility of applying a plasmonic nanopore to DNA and protein detection, which may also provide an easy way for tracking single molecule translocation by developing a well-defined single plasmonic nanopore.