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The content of chlorophyll existed as an indicator in plant leaves plays a vital role in plant growth.An experimental setup using a three-wavelength diffuse reflectance was proposed.The LEDs with the peak wavelengths of 640 nm, 660 nm and 940nm are utilized to measure the total chlorophyll content.Two different maize breeds, Zhengdan-958 and Xundan-20, which were fertilized in different levels before jointing stage, were verified by using this setup.The regression analyses between remission function values of diffuse reflectance and SPAD values, remission function values of diffuse reflectance and the real content of total chlorophyll were established.The correlation coefficients of R square between remission function values and the real content of total chlorophyll were 0.9766 for the Zhengdan-958 leaves and 0.9612 for the Xundan-20 leaves.The experimental results validated the feasibility of diffuse reflectance spectrum for the detection of the total content chlorophyll.It also provides the guidance for the development of the portable instrument to determine the real chlorophyll content.