The invention discloses a method for separating L-phosphinothricin and gluconic acid by using ion exchange resin. The method comprises the steps: selecting a crude L-phosphinothricin solution containing L-phosphinothricin and gluconic acid, and regulating the pH value of the crude solution to 1-4 to obtain a feeding solution; adding the feeding solution into an exchange column filled with strong-acid anion exchange resin, carrying out chromatographic separation, and collecting effluent containing gluconic acid; stopping adding the feeding solution when detecting that the concentration of L-phosphinothricin reaches 5-10% of the concentration of L-phosphinothricin in the feeding solution at the bottom of the exchange column; and then, washing the resin by using deionized water, desorbing L-phosphinothricin by using an alkaline solution serving as an eluent, and collecting the eluant to obtain an L-phosphinothricin solution. According to the method, the pH value of the crude L-phosphinothricin solution is adjusted firstly, and then, chromatographic separation is performed by using the strong-acid anion exchange resin, so that the efficient separation of L-phosphinothricin and gluconicacid is realized, and the separation efficiency is high.