The effect of illumination intensity on automatic focusing in the microscopic vision research field is discussed.An automatic illumination control system is developed to provide experimental conditions for illumination research on microscopic vision.The fast Fourier transform(FFT) is used to analyze spectra of image sequences at defocus and image sequences under different illumination intensities,which proves that illumination intensity conditions impact significantly on the high-frequency energy of images.A evaluating function for the high-frequency energy of images is proposed.The experimental results show that illumination intensity has a great impact on focusing curves,and higher accuracy of 0.857 μm can be achieved after illumination optimization,which shows that illumination opitimization can improve automatic focusing accuracy.