Through carrying out spectral test experiment, the influence factors of spectrum test were analyzed, the influence degree of various factors in spectral recognition was explicated and the method of spectra test was optimized for cotton leaf infected by verticillium wilt. The results indicated that under different severity levels, the shape and value of reflectance of disease symptoms part were Significantly higher than healthy part on cotton leaf, compared with the black board as baseboard, the spectral values of disease leaves were slightly higher in visible light wavebands and significantly higher in others wavebands than healthy leaves on white baseboard. Different position of leaf on cotton plant has different effect degree to the recognition of disease, the effect of stem leaf was more obvious than that of else leaf, the identical leaf position was less influenced by disease than that of others. The effect of healthy leaf was smaller than disease leaf. The reflectance of leaf back was higher than front in visible light waveband, from high to flat, and then low in near infrared waveband, and from high to low to in short infrared waveband. Test time and cotton varieties had less influence on recognizing disease by spectra, and the effect of the same condition was acceptable. Test site had no effect on disease recognition by spectra. The effect of each factor was different for recognizing disease leaf by spectra, and this study will provide reference for the researchers of crop disease diagnosis by spectra.