We designed peanut agglutinin (PNA)-immobilized fluorescent nanospheres as a non-absorbable endoscopic imaging agent capable of being administered intracolonically. Following our previous researches with evidence that the imaging agent recognized small-sized colorectal tumors on the mucosal surface with high affinity and specificity in animal experiments, a potential of this nanoprobe as a drug candidate was evaluated from a safety perspective. The imaging agent detects colorectal tumors through recognition of the tumor-specific antigen by PNA immobilized on the nanosphere surface, and the detection is made via the fluorescent signal derived from coumarin 6 encapsulated into the nanosphere core. The stability studies revealed that the high activity of PNA was maintained and there was no significant leakage of coumarin 6 after intracolonic administration of the imaging agent. Cytotoxicity studies indicated that no local damage to the large intestinal membrane was induced by the imaging agent. Further, in vitro and in vivo permeation studies demonstrated that there was no significant permeation of the imaging agent through the monolayer of cultured cells and that the imaging agent administered locally to the luminal side of the large intestine was almost completely recovered from the administration site. Therefore, we concluded that the imaging agent is a safe and stable probe which remains in the large intestine without systemic exposure.