The image quality and resolution will be affected, if the multiplication gain value of EMCCD imaging system is too low or too high. This paper presents the algorithm of EMCCD automatic gain adjustment based on fixed gray level. The algorithm takes the average brightness of the image as a measure of image quality. It calculates the multiplication gain adjustment value through the calculation of average brightness of current frame image, combining the two gray level threshold values and the system exposure function, so that the next frame image will achieve the ideal brightness value. On the basis of the algorithm, this paper builds a multiplication gain adjustment control circuit and a multiplication gain resistor lookup table. Then, we can achieve automatic adjustment of multiplication gain by changing the resistance value of the digital potentiometer in the circuit. Experimental results show that the image quality can be effectively improved, by using the algorithm of automatic gain adjustment based on fixed gray level. After adjustment, the brightness of the image is moderate, contrast enhanced, and the details more clear.