The invention provides a separating and purifying method of L-isoleucine, and belongs to the field of bioengineering. The method comprises the following steps of (1) fermentation liquor purification,namely adding an ion removing agent into L-isoleucine fermentation liquor, and conducting centrifugal treatment on a mixture after heating the mixture to obtain supernate; (2) primary purification, namely conducting ultrafiltration treatment on the supernate obtained in the step (1) through ultrafiltration membrane to obtain clear filter liquor; (3) high purification, namely conducting acidizing treatment on the clear filter liquor obtained in the step (2), then adding 001*7-type strong acidic styrene cation exchange resin for dynamic adsorption, then using eluent for elution, and obtaining eluant; and (4) crystallization, namely adjusting a pH value of the eluant obtained in the step (3) to be 7.5, adding ethyl alcohol for crystallization, and obtaining the L-isoleucine after conducting vacuum drying on crystals. By means of the separating and purifying method of the L-isoleucine, impurities in the fermentation liquor can be effectively removed, and the extraction rate and the qualityof the L-isoleucine are improved so that the separating and purifying method of the L-isoleucine can be widely applied in industrial production of L-isoleucine.