Abstract: The influences of the roughness of fracture surface and the loading-unloading environment on seepage characteristics of fracture cannot be ignored. To study the influences of the fracture surface with different joint roughness coefficients (JRC) and loading-unloading of confining and osmotic pressures on the seepage evolution laws of rough single fracture of limestone, five groups of limestone samples with a single fracture with different JRCs are prepared. The self-developed seepage test system for the fractured rock mass is used to conduct a seepage test of the fractured samples with different JRCs under loading-unloading of confining and osmotic pressures. The results show that the seepage flow and permeability of different fractured samples decrease with the increase of the confining pressure, and the permeability of each sample has a power function relationship with the confining pressure, with the minimum R² of 0.9583. There is an obvious hysteretic effect during the unloading of the confining pressure. The JRC has a great influence on the hysteretic degree during unloading, which is logarithmic with the decline and recovery. During the process of loading-unloading of pressure, the change of seepage flow through the single fracture is positively correlated with the osmotic pressure, while the permeability decreases continuously. There is a good correlation between the decreased value of seepage flow through the single fracture and the JRC after a cycle of loading-unloading of pressure. A prediction model for seepage flow of fracture after loading-unloading of osmotic pressure is proposed.