Nep detection system used in carding machine is a kind of new on-line detection system based on machine vision. The function of light source is illumination. Light source is an important factor for cleaning rate. The light source system should be optimized and designed to satisfy the broad surface, high speed and high sensitivity for on-line nep detection system. By experiments, we could know that the neps in cotton web would have the highest reflectivity illuminated by which kind of light source. Synchronously, we could know that the neps and surrounding structure how to get the best contrast. Compared with the spectral curve of traditional fluorescence lamp, high pressure mercury lamp and LED, the scheme of combination in He-Ne laser and optical system was adopted in this paper. Experimental results show that this light source, for the circumstance of nep detection in carding machine, is better than other light sources because of the high brightness and straightness of laser.