Hydroxamamide (Ham) is a thiol-free chelating agent that forms technetium-99m (99mTc)-complexes with a metal-to-ligand ratio of 1:2 under moderate reaction conditions. Therefore, Ham-based chelating agents will produce 99mTc-labeled compounds with a bivalent targeting scaffold. For their universal usage, we developed a novel Ham-based bifunctional chelating agent, "Ham-Mal", with a maleimide group that can easily conjugate with a thiol group, for to preparing 99mTc-labeled bivalent ligand probes. Ham-Mal was synthesized by a four-step reaction, and then reacted with cysteine or c(RGDfC) to produce Ham-Cys or Ham-RGD. These precursors were reacted with 99mTcO4- for 10 min under room temperature to obtain 99mTc-(Ham-Cys)2 and 99mTc -(Ham-RGD)2. The cellular uptake level of 99mTc-(Ham-RGD)2 by U87MG (high Integrin ɑvβ3 expression) cells was significantly higher than that by PC3 (low Integrin ɑvβ3 expression) cells at 60 min after the incubation, and the uptake was significantly suppressed by pre-treatment for 15 min with excess c(RGDfK) peptide. In the in vivo study with U87MG/PC3 dual xenografted BALB/c-nu mice, the radioactivity of U87MG tumor tissue was significantly higher than that of PC3 tumor tissue at 360 min after the administration of 99mTc-(Ham-RGD)2. These results suggest Ham-Mal may have potential as a bifunctional chelating agent for 99mTc-labeled bivalent ligand probes.