This paper regards the dynamic performance of radio frequency identification (RFID) positioning system as the analysis target. In this paper, the transmission from the static performance evaluation of RFID positioning system to the dynamic one is done. The Fisher information matrix and identification value are introduced to evaluate the dynamic positioning performance of RFID system. The positioning performance at the case of different paths, motion rates and number of reference tags could be evaluated by calculating the Fisher information matrix and identification value. The numerical simulation shows that motion paths and rate of target and the number of reference tags, could directly affect the positioning performance of RFID system. The findings also indicate that the selected motion path and rate of the target have a direct impact on the positioning performance. Furthermore, adjusting motion path of target close to the geometric center of reference tags and reducing motion rate and acceleration appropriately could optimize the dynamic identification performance of RFID positioning system. This method provides a reference for improving the positioning performance and reducing the measurement error of RFID system