Phage display technique provides a powerful approach for the discovery of new tumor-specific peptides. However, the peptides isolated through this technique usually did not possess high tumor-specific property. A pre-clearing step was introduced to increase the efficiency of biopanning by removal of particles that could interact with ubiquitously expressed cellular receptors in the non-target organs. The randomized Ph.D-CX₇C phage library (Phage III) was first pre-cleared in normal mice to reduce vasculature- or organ-targeting phages to get the pre-cleared phage library, and then the tumor-targeting bacteriophage particles (Phage I) were screened from pre-clearing phage library in S180 tumor-bearing mice. The biodistribution results of ^99mTc-labeled phages in mice bearing S180 tumor show that the uptake of ^99mTc-labeled Phage I in tumor is high but low in normal organs, and the tumor-to-liver and tumor-to-spleen ratios of ^99mTc-labeled Phage I are higher than those of ^99mTc-labeled Phage II (tumor-specific phages screened from the original CX7C library) and Phage III (unscreened phages from the original CX7C library). It indicates that the yield of tumor-targeting bacteriophage particles could be improved and the non-specific binding in organs becomes weak. Consequently, the pre-clearing phage display method could improve the yield of positive hits by reducing the non-target organ accumulation of bacteriophage particles.