Abstract: Growth circle method is a simple, convenient, economical and effective way to preliminarily screen bacteria with high L-isoleucine output.In this paper, some factors including the temperature of test culture medium when the directional bacteria were added to, the mass of directional bacteria, agar concentration of test plate and agar concentration of agar pieces which affected measurement by bacterial growth circle method were studied.The optimal method was given as follows:minimal medium was chosen for test plate and agar pieces, the upper and lower parts of test plate were 10mL mixed bacteria culture medium and 10mL minimal medium, separately and the concentration of agar was 1%, the concentration of agar in agar pieces was 1.5%, the incubation time, the amount optimal temperature of directional bacteria in mixed bacteria plate were 18h, 4% and 55℃, respectively.