Abstract: In order to develop an ideal ⁹⁹Tc^m-labeled glucose metabolism imaging agent, MAMA-conjugated azadibenzocyclooctyne derivative and four 2-azido glucose derivatives were synthesized. Two potential candidates (N₃-DG-1 and N₃-DG-2) were selected according to the in vitro kinetic study experiment. The 2-azido glucose derivatives were pretargeted in mice bearing S180 tumor, then ⁹⁹Tc^m-labeled MAMA-azadibenzocyclooctyne derivative was administrated to accomplish the in vivo copper-free click reaction and imaging of glucose metabolism. The biodistribution results showed that the tumor/blood and tumor/muscle ratios of N₃-DG-2 were higher than those of N₃-DG-1, indicating the combination of ⁹⁹Tc^m-labeled MAMA-azadibenzocyclooctyne derivative and N₃-DG-2 was more suitable for glucose metabolism imaging.