Purpose: Current skin imaging modalities, including optical, electron, and confocal microscopy, mostly require tissue fixations that could damage proteins and biological molecules. Live tissue or cell imaging such as ultrasonography and optical coherent microscope may not adequately measure the dynamic spectroscopical changes. Raman spectroscopy has been adopted for skin imaging in vivo, mostly for skin cancer imaging. However, whether the epidermal and dermal thickening in skin could be measured and distinguished by conventional Ramen spectroscopy or the surface-enhanced Raman scattering (SERS), a rapid and label-free method for noninvasive measurement remains unknown.Methods: Human skin sections from patients of atopic dermatitis and keloid, which represent epidermal and dermal thickening, respectively, were measured by conventional Ramen spectroscopy. In mice, skin sections from imiquimod (IMQ)-and bleomycin (BLE)-treated mice, which reflect the epidermal and dermal thickening, respectively, were measured by SERS, that incorporates gold nanoparticles to generate surface plasma and enhance Raman signals.Results: Conventional Ramen spectroscopy failed to consistently show the Raman shift in human samples among the different groups. SERS successfully revealed a prominent peak around 1300 cm −1 in the IMQ-treated skin; and two significant peaks around 1100 and 1300 cm −1 in BLE-treated group. Further quantitative analysis showed 1100 cm −1 peak was significantly accentuated in the BLE-treated skin than that in control skin. SERS identified in vitro a similar 1100 cm −1 peak in solutions of collagen, the major dermal biological molecules. Conclusion: SERS distinguishes the epidermal or dermal thickening in mouse skinwith rapid and label-free measures. A prominent 1100 cm −1 SERS peak in the # These two authors contributed equally to this study.