Phage display technique is a powerful approach for discovering new tumor- and organ-targeting ligands, and radiolabeled phage has a potential to analyze the phage-binding sensitivity and specific imaging. In this study, phage II (the spleen-targeting phage) in mice was isolated after three rounds biopanning, and labeled by ^99mTc using mercaptoacetyltriglycine (MAG₃) as chelator to evaluate their binding properties in vivo. The amount of phage II eluted from spleen was enriched by plague assay each round. ^99mTc-MAG₃-phage II showed the less retention in blood at any time point than half that of ^99mTc-MAG₃-phage I (the radiolabeled original Ph.D-12 phage as control). The accumulation in spleen between ^99mTc-MAG₃-phage I and II was of different tendency. The highest uptake of ^99mTc- MAG₃-phage II in spleen was 24.80 %ID/g at 30 min; and of ^99mTc-MAG₃-phage I, 30.93% ID/g at 5 min. After circulating ^99mTc-MAG₃-phage II for 120 min, its accumulation in spleen decreased though higher than that of ^99mTc- MAG₃-phage I. In other organs, the ^99mTc-MAG₃-phage II showed low retention and high spleen-to-organ or tissue ratios. In conclusion, the radiolabeled phage II is convenient for studying the binding and specificity of spleen- targeting peptides found via phage display in vivo.