Introduction 99mTc-labeled hexavalent probes can be readily synthesized by the coordination of six equivalent isocyanide ligands towards TcI, and alkyl isocyanide ligands have been extensively used for preparing such probes. However, high ligand concentration (>1 mM) is generally required due to their insufficient coordination ability to TcI. Methods and results In this study, we revealed that aryl isocyanide ligands, which have greater π-accepting ability compared with alkyl ones, provided 99mTc-labeled hexavalent probes in high radiochemical yields (>95%) even at low ligand concentration (50 μM). We applied this finding to the synthesis of a 99mTc-labeled hexavalent RGD probe, targeting integrin αvβ3. This 99mTc-labeled probe was prepared in a 5 min reaction at ligand concentration of 50 μM, and exhibited high tumor localization in vivo without post-labeling purification. Conclusion The present findings indicate that aryl isocyanide ligands would be a useful precursor to a variety of 99mTc-labeled hexavalent targeting probes for molecular imaging of saturable systems. Advances in knowledge Aryl isocyanide is a better precursor than alkyl isocyanide for preparing 99mTc-labeled hexavalent targeting probe. Implication for patient care This work provides a straightforward method to prepare molecular imaging agents of high target uptake, which would facilitate nuclear medicine imaging in clinical settings.