This study aims to evaluate the composition, micro-structure and inorganic phase alternations of human dentine irradiated by Er,Cr:YSGG laser with water cooling spray system. X-ray diffraction (XRD) analysis indicated that the main inorganic phase of dentine before and after laser irradiation were all Hydroxyapatite (HA) structure, approximately 30 nm in size. No significant changes occurred in the average particle size after irradiation in four energy densities (6.18 J/cm2, 8.04 J/cm2, 9.89 J/cm2, 11.1 J/cm2). Atomic force microscope (AFM) phase image and the energy-dispersive spectroscope (EDS) analysis, however, demonstrated that the thermal effects of Er,Cr:YSGG laser with water-cooling spray system on the dentin surface was intense enough to induce notable decrease of the organic matter. Both scanning electron microscopy (SEM) and AFM analysis showed that the irradiated dentine presented rough surface morphology. The surface is clean and dentinal tubules are completely open. The ablation rate of both peri- and intertubular dentine increased at higher energy densities but no significant changes of gross appearance took place. Chemical analysis reveals that laser photothermal effect would decrease significantly the organic content of superficial dentinal layer. We conclude that the Er,Cr:YSGG laser, as a new type clinic laser, would not significantly influence the inorganic phase structure of the surface dentine layer, however, thermal ablation was occurred in organic component. Moreover, the rough ablated surface as well as the opened dentinal tubules induced by irradiation, might be advantageous to the infiltration of the adhesive materials, thus the adhesion of dental restoration could be enhanced. Further studies should focus on the correlation between bond strength and Er,Cr:YSGG lased dentine.