The invention discloses a method for separating a Casparian strip, which comprises the following steps that: 1) a plant primary root is soaked with a segregating liquid, and the soaked plant primary root is recorded as a segregated plant primary root; 2) the segregated plant primary root is enzymolyzed, and the segregated plant primary root is recorded as an enzymolyzed plant primary root; and 3)the enzymolyzed plant primary root is separated to obtain the Casparian strip, wherein the segregating liquid is of an isometric liquid mixture of (7-12)% ( mass percentage) of aqueous chromic acid solution and (7-12)% (mass percentage) of aqueous nitric acid solution. The invention has the following advantages: 1) the operation is simple and fast, and the separation time is shortened; 2) cellulase and pectinase are used for moderate enzymolysis, so the use amount of enzyme is reduced, and the cost is saved; and 3) the separation effect is good.