The research was focused on the separation of single hemp (Cannabis sativa L.) fibre cells with low fineness from mechanically extracted fibre bundles of high fineness. The fiber bundles were treated with enzymes, namely panzym, pectinase, hemicellulase, and cellulase, along with a combination of panzym and ultrasonic treatments. Changes in the fiber structure were followed at molecular and microscopic levels by means of NIR FT Raman spectroscopy and Environmental Scanning Electron Microscopy (ESEM). Buffer-panzym treatments of hemp fibers had a prominent effect in loosening of the fiber cells. The best of refining was achieved when the fiber bundles were treated with buffer-panzym solution in combination with ultrasonic treatment.