A gold nanoparticle (AuNP) has a localized surface plasmon resonance peak depending on its size, which is often utilized for surface-enhanced Raman scattering (SERS). To obtain information on the cholesterol (Chol)-incorporated lipid membranes by SERS, AuNPs (5, 100 nm) were first functionalized by 1-octanethiol and then modified by lipids (AuNP@lipid). In membrane surface-enhanced Raman spectroscopy (MSERS), both signals from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Chol molecules were enhanced, depending on preparation conditions (size of AuNPs and lipid/AuNP ratio). The enhancement factors (EFs) were calculated to estimate the efficiency of AuNPs on Raman enhancement. The size of AuNP100nm@lipid was 152.0 ± 12.8 nm, which showed an surface enhancement Raman spectrum with an EF2850 value of 111 ± 9. The size of AuNP5nm@lipid prepared with a lipid/AuNP ratio of 1.38 × 104 (lipid molecule/particle) was 275.3 ± 20.2 nm, which showed the highest enhancement with an EF2850 value of 131 ± 21. On the basis of fluorescent probe analyses, the membrane fluidity and polarity of AuNP@lipid were almost similar to DOPC/Chol liposome, indicating an intact membrane of DOPC/Chol after modification with AuNPs. Finally, the membrane properties of AuNP@lipid systems were also discussed on the basis of the obtained MSERS signals.