Abstract: An accurate and efficient Dynamic Framed-Slotted ALOHA (DFSA) anti-collision algorithm is proposed based on unequal timeslots in Radio Frequency IDentification (RFID) system. Considering the influence of idle and collided timeslots on RFID system efficiency, the algorithm adopts an optimized DFSA anti-collision strategy based on unequal timeslots, which determines frame length by optimized parameter and unread tags, performs tag estimation with optimized Chebyshevs inequality, and analyses the process of identification based on Markovian for controling read cycles. The analysis and simulations show that the proposed algorithm achieves better system performance, reduces identification time, and has higher accuracy than Low Bound (LB), Schoute and Cratio estimations.