A flexible hybrid substrate was developed by affixing gold nanoparticles (AuNPs) onto the surface of two-dimensional nanomica platelets (NMPs). The substrate was successfully used in biosensors with high efficiency and high selectivity through surface-enhanced Raman scattering (SERS). By controlling the amphiphilicity of the hybrid substrate, the flexible substrate was made highly selective toward biomolecules. Four different SERS substrate systems were constructed, including intercalated mica, exfoliated NMPs, hydrophilic exfoliated NMPs, and hydrophobic exfoliated NMPs. NMPs were only 1 nm thick. AuNPs adsorbed on both sides of NMPs and thus created excellent three-dimensional hot junction effects in the z-axis direction. For the detection of adenine in DNA, a satisfactory Raman enhancement factor (EF) of up to 8.9 × 106was achieved with the detection limit as low as 10-8M. Subsequently, the AuNP/NMP hybrids were adopted to rapidly detect hydrophilic Staphylococcus hominis and hydrophobic Escherichia coli. The AuNP/PIB-POE-PIB/NMP nanohybrid was concurrently hydrophilic and hydrophobic. This amphiphilic property greatly enhanced the detection selectivity and signal intensity for hydrophilic or hydrophobic bacteria. Overall, AuNPs/PIB-POE-PIB/NMPs developed as SERS substrates enable rapid, sensitive biodetection.