It was demonstrated that hydrophobicity of solid supported planar dipalmitoyl phosphatidylcholine (DPPC) phospholipid multilayer can be greatly increased by incorporating a transmembrane protein, gramicidin, into the DPPC membrane. The contact angle of deionized water droplet on the gramicidin-modified DPPC membrane increased from 0 degrees (complete wetting) without gramicidin to 55 degrees after adding 15 mol % gramicidin. The increased hydrophobicity of the gramicidin-modified DPPC membrane allowed the membrane to remain stable at the air/water interface as well as underwater. The Au nanoparticles deposited on the gramicidin-modified DPPC membrane reproduced the characteristic surface plasmon resonance peak after being kept underwater or in phosphate-buffered saline solution for 5 days, attesting to the membrane stability in an aqueous environment. The enhanced underwater stability of the lipid multilayer substantially broadens the potential application of the lipid multilayer which includes biosensing, enzymatic fuel cell, surface enhanced Raman spectroscopy substrate.