The resistance of nanoporous dentin biomaterial to CO2 laser irradiation was investigated by experiment and simulation for potential tooth hypersensitivity treatment. The controlled parameters were laser power of 0.03-0.150 W, scanning speeds of 11.4-34.2 mm/s and focus/defocus modes for studying interaction between laser energy and dentin of human tooth. Most of dentin specimens were etched after CO2 laser irradiation at the power larger than 0.075 W and the scanning speed of 11.4 mm/s. Compared with simulation results of temperature distribution, maximum surface temperature of those etched specimens are between 1961 and 3127 °C which exceeded the melting point (1570 °C) of dentin's main content of hydroxyapatite (HA). Increasing scanning speed can reduce the linear density of laser output energy for just locally melting porous microstructure of dentin surface without etching. Varying focus mode can also improve the damage of nanoporous dentin microstructure. At parameters of 0.150 W power and 34.2 mm/s scanning speed under defocus operation, laser treatment was successful performed on the nano-HA coated dentin with well-molten sealing on tubules of porous microstructure at a simulate surface temperature about 574 °C, which was potential for dentin hypersensitivity cure application.