Two novel cyanine-based 1-amino-1-deoxy-beta-glucose conjugates (Glu-1N-Cy3 and Glu-1N-Cy5) were designed, synthesized and their fluorescence characteristics were studied. Both Giu-1N-Cy3 and Glu-1N-Cy5 accumulate in living HT29 human colon cancer cells, which overexpress glucose transporters (GLUTs). The cellular uptake of the bioprobes was inhibited by natural GLUT substrate D-glucose and 2-deoxy-D-glucose. The GLUT specificity of the probes was validated with quercetin, which is both a per meant substrate via GLUTs and a high-affinity inhibitor of GLUT-mediated glucose transport. Competitive fluorometric assay for GLUT substrate cell uptake revealed that Glu-1N-Cy3 and Glu-1N-Cy5 are 5 and 10 times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. This study provides fundamental data supporting the potential of these two conjugates as new powerful tools for GLUT-mediated theranostics, in vitro and in vivo molecular bioimaging and drug R&D. (C) 2016 Elsevier Inc. All rights reserved.