In a passive Ultra-High Frequency (UHF) Radio Frequency IDentification (RFID) system, when multiple tags choose a same time slot to send information to a reader, tag collision will occur. Generally, the collision is resolved only on a Medium Access Control (MAC) layer. In fact, the collision could be separated on a physical layer, and the efficiency of system identification could be advanced. In the physical layer separation, channel estimation is one of key techniques because good estimation could help to correctly recover the collided signals. Conventional channel estimates work well under the environment of two collided tags. When the number of collided tags is beyond two, however, the conventional channel estimates have more estimation errors. In this paper, a novel channel estimate method is proposed for the passive UHF RFID signal separation on physical layer. The proposed method uses the information of preambles which is a-priori known for a reader and applies a Least-Square (LS) criterion to estimate the channel parameters. From numerical results, the estimation errors of the proposed method are lower than the conventional methods under the number of collided tags is more than two. And, the separation efficiency of the proposed methods is also higher.