High gradient magnetic separation (HGMS) is effective for the separation of weakly magnetic minerals, and this method is achieved through the use of matrix, which is made of huge numbers of rod wires. So that the coupling effect of magnetic field and flow field between wires has a marked effect on the HGMS performance. In the investigation, the coupling effect between magnetic wires and its influence on high gradient magnetic separation performance were theoretically described and simulated using COMSOL Multiphysics. It is found that the magnetic field round a wire would be affected by the neighboring wires, and then a coupling effect of magnetic field between wires was produced, increasing the magnetic induction intensity on the upstream and downstream of wire surface. And the coupling effect of flow field could increase the slurry velocity at the regions of the wire surface with azimuth angles of 0° and 90°, which is beneficial for the selective capture of wire. These simulated results were basically validated with the experimental separation, using an innovative Magnetic Capture Analysis Method. It is found that the wire spacing has significant effect on the coupling effect of magnetic wires, and a critical spacing for wires could achieve an excellent coupling effect, which is beneficial for the improvement of HGMS performance. This investigation contributes to improve HGMS performance in concentrating fine weakly magnetic ores.