Roughness of rock fracture has a significant influence on the seepage characteristics of fracture. The point cloud data of rock fracture surface was collected by three-dimensional (3D) optical scanning system. The joint roughness coefficient (JRC) and surface roughness ratio (R_s) were calculated by SURFER and GEOMAGIC STUDIO, and the quantitative relationship between JRC and Rs was established. The effects of JRC and R_s on the fracture seepage characteristics in limestone were investigated by conducting seepage tests under the coupling effect of stress, seepage and chemical interaction. The results show that JRC is a logarithmic function of R_s, with the R-squared (R²) of 0.912 8. The maximum relative error (MRE), mean absolute error (MAE) and root mean square error (RMSE) between the proposed characterization formula and seepage test results are 6.93%, 0.34 and 0.27, respectively. JRC shows quadratic function and logarithmic function with the seepage flow and permeability at stable period, respectively. The fitted relationships of Rs and each parameter are consistent with that of JRC. The larger JRC is, the smaller the seepage flow and permeability are. The values of JRC and R_s of the fracture surface increase under the coupling effect of HMC (hydrological- mechanical-chemical) three fields. The characterization method proposed in this study can be used to estimate the surface roughness of rock fracture. Meanwhile, the value of JRC can be applied to predict the seepage flow and the permeability at stable seepage period.