Wool fibers are composed of cuticle and cortex cells, which are of obvious differences in many properties. The development of methods to isolate the two kinds of cells can provide platform to elucidate the roles they play in the characteristics of wool fibers. Here we demonstrate the cuticle can be completely and rapidly removed from the wool fibers by the use of ionic liquids, with inner cortex intact. Confocal microscope, SEM and FTIR have been applied to study the wool fibers after cuticle removal. In contrast to the traditional/long physical or chemical separation routes (>14 h), our method is very rapid (<1 h). This work demonstrates the ability of ionic liquid as a novel, rapid and efficient media for cuticle/ cortex isolation.