Aiming at the problems of conventional Radio Frequency Identification(RFID) positioning process, Differential Evolution(DE) algorithm is proposed to optimize the positioning accuracy of RFID. This method first randomly initializes reference tag location coordinates, calculates the distance between reader and tag by using Received Signal Strength(RSS) value. Then it estimates the location coordinates of the nearest label under test by optimizing the reader and the distance error between the reference labels and tags under test. Finally, it is compared with classical LANDMARC positioning systems. The simulation results show that the average positioning error of the classical LANDMARC positioning system is 1.115 8 m, and that of the system optimized by DE algorithm is 0.001 2 m, which proves that the method of RFID positioning optimized by DE algorithm is effective.