Concanavalin A (ConA), which exists in jack bean, can specifically bind to either mannose or glucose units. Given that ConA is a potential anticancer agent for antihepatoma therapy, analytical methods that can be used for ConA detection in plant extracts or biological fluids are important. Labeling agents that can be used to distinguish cancer cells from noncancer cells are also useful for cancer diagnostics. In this study, maltose-directed synthesis of Au nanoparticles (NPs; Au@Maltose NPs) were generated from one-pot reactions and used as sensing probes toward ConA tetrameric forms. Results showed that the binding affinity between Au@Maltose NPs and ConA was desirable with a dissociation constant as low as ∼6.66 × 10-8 M. Moreover, the limit of detection against ConA was estimated to be ∼23 pM, which was the lowest ever reported when Au NPs were used as sensing probes against ConA. Moreover, Au@Maltose NPs were used as imaging agents for ConA-treated cancer cells, such as human hepatoma HepG2 cells and human breast T-47D cancer cells, which can overexpress mannose N-glycan units on the cell membrane. NIH 3T3 fibroblast cells, which are noncancer cells, were used for comparison. Results demonstrated that the imaging agent made from combined ConA and Au@Maltose NPs can be used to distinguish cancer cells from noncancer cells.