Binding modes of a dibenzotetraaza[14]annulene (DBTAA) derivative with synthetic nucleic acids were studied using surface-enhanced Raman spectroscopy (SERS). Changes in SERS intensity and appearance of new bands in spectra were attributed to different complexes formed between the DBTAA molecules and DNA/RNA polynucleotides. A decrease in intensity pointed to intercalation as the dominant binding mode of the annulene derivative with poly dGdC-poly dGdC and poly rA-poly rU, whereas new bands in the spectra at 735 cm−1 and 1345 cm−1 revealed binding within the minor groove of poly dAdT-poly dAdT. When all the dominant binding sites were occupied, SERS spectra implied that small molecules bind on the outside of the DNA analogues, while exist mainly as free molecules in equimolar ratio with the synthetic RNA polynucleotide, thereby indicating higher affinity for DNA than for RNA.