Abstract: Transmission colorimetric method is a traditional method of chlorophyll content nondestructive determination.Plant leaves thickness significantly affect the accuracy of measurement.In this paper, an experimental instrument using a three-wavelength diffuse reflectance was proposed.Three different maize breeds, Zhengdan-958, Xundan-20 and Yudan-606 were verified by using this instrument.The regression analyses between remission function values of diffuse reflectance and the real content of total chlorophyll were established.The determination coefficients of R2 between remission function values and the real content of total chlorophyll were 0.9766 for the Zhengdan-958 leaves, 0.9612 for the Xundan-20 leaves and 0.9409 for the Yudan-606 leaves.The experimental results showed the feasibility of diffuse reflectance spectrum for the detection of the total content chlorophyll.