The gold-assisted exfoliation is a very effective method to produce large-area (cm2-scale) membranes of molybdenum disulfide (MoS2) for electronics. However, the strong MoS2/Au interaction, beneficial for the exfoliation process, has a strong impact on the vibrational and light emission properties of MoS2. Here, we report an atomic force microscopy, micro-Raman, and micro-photoluminescence (μ-PL) investigation of 2H-MoS2 with variable thickness exfoliated on Au and subsequently transferred on an Al2O3/Si substrate. The E2g–A1g vibrational mode separation Δω (typically used to estimate MoS2 thickness) exhibits an anomalous large value (Δω ≈ 21.2 cm−1) for monolayer (1L) MoS2 on Au as compared to the typical one (Δω ≈ 18.5 cm−1) measured on 1L MoS2 on Al2O3. Such substrate-related differences, explained in terms of tensile strain and p-type doping arising from the MoS2/Au interaction, were …