In the synthesis of technetium-99m (99mTc) labeled target-specific ligands, the presence of a large excess of unlabeled ligands over 99mTc in the injectate hinders target accumulation of 99mTc-labeled ligands by competing for target molecules. To circumvent the problem, we recently developed a concept of the metal coordination-mediated multivalency, and proved the concept with a 99mTc-labeled trivalent compound [99mTc(CO)3(CN-RGD)3]+. In this study, D-penicillamine (Pen) was selected as a chelating molecule and a cyclic RGDfK peptide was conjugated to Pen via a hexanoic linkage (Pen-Ahx-c(RGDfK)). 99mTc complexation reaction, and the stability, integrin αvβ3 binding affinity, and biodistribution of the 99mTc-labeled probe were investigated to evaluate the applicability of the concept to bivalent probes. 99mTc-[Pen-Ahx-c(RGDfK)]2 was obtained over 95% radiochemical yields under low Pen-Ahx-c(RGDfK) concentration (50 μM). 99mTc-[Pen-Ahx-c(RGDfK)]2 showed approximately 10-times higher integrin αvβ3 binding affinity than the monovalent compounds, Pen-Ahx-c(RGDfK) and c(RGDyV). In biodistribution studies, the tumor accumulation of 99mTc-[Pen-Ahx-c(RGDfK)]2 was decreased to 77% and 43% of HPLC-purified (Pen-Ahx-c(RGDfK)-free) 99mTc-[Pen-Ahx-c(RGDfK)]2 by the presence of 5 nmol of unlabeled Pen-Ahx-c(RGDfK) and Re-[Pen-Ahx-c(RGDfK)]2, respectively. 99mTc-[Pen-Ahx-c(RGDfK)]2 provided tumor image without removing unlabeled ligand, while a 99mTc-labeled monovalent probe prepared from a monovalent ligand could not. These findings indicate the availability of the design concept to prepare 99mTc-labeled bivalent probes with a variety of 99mTc core and other metallic radionuclides of clinical relevance.