Peanut agglutinin (PNA)-immobilized polystyrene nanospheres with surface poly(N-vinylacetamide) (PNVA) chains encapsulating coumarin 6 were designed as a novel colonoscopic imaging agent. PNA was a targeting moiety that binds to beta-D-galactosyl-(1-3)-N-acetyl-D-galactosamine, which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. PNVA was immobilized with the aim of reducing nonspecific interactions between imaging agents and normal tissues. Coumarin 6 was encapsulated into nanosphere cores to provide endoscopically detectable fluorescence intensity. After incubation of imaging agents with human cells, the fluorescence intensity of imaging agent-bound cells was estimated quantitatively. The average fluorescence intensity of any type of colorectal cancer cell used in this study was higher than that of small intestinal epithelial cells that had not exposed the carbohydrate. The in vivo performance of imaging agents was subsequently evaluated using a human colorectal cancer orthotopic animal model. Imaging agent-derived strong fluorescence was observed at several sites of the large intestinal mucosa in the tumor-implanted nude mice after the luminal side of the colonic loop was contacted with imaging agents. In contrast, when mice that did not undergo tumor implantation were used, the fluorescence intensity on the mucosal surface was extremely low. Data indicated that imaging agents bound to colorectal cancer cells and the cancer cell-derived tumors with high affinity and specificity.