After acquiring the ultraviolet absorption spectrum and its first order derivative spectrum distributions of Avermectin B1 by using UV-2401 PC spectrophotometer and spectrum-measuring system, we found that coordinates of the peak A252.2 and the valley A256.8 are not disturbed by the different forms of preparations. We considered that delta A = A252.2-A256.8 is the reliable ground for quantitative analysis of Avermectin B1 content in order to reduce error. The standard regression equation c = 272.7 delta A-0.161 and the related coefficient r = 0.9996 are obtained through measurements of the standard preparations. Then the Avermectin B1 content and the average recovery rate of different preparations are determined and the result indicates that this method is especially suitable for determining the Avermectin B1 content.