The intraoperative precision cleaning of lymph nodes (LNs) is an essential component of treating neoplastic disease. To develop efficient probes for the targeted detection of LNs that could act as carriers for the specific diagnosis and treatment of metastatic LNs in the future, dextran-coated iron oxide nanoclusters (DIONs) were synthesized using a one-pot coprecipitation procedure. These modified DIONs have good water dispersibility, cytocompatibility, an optimum size, and a stable, dark brown color for LN imaging. In this study, cytotoxicity was evaluated using lymphatic endothelial cells (LECs) to predict biosafety and biocompatibility. Most importantly, the effectiveness of DIONs in mapping perigastric LNs in Sprague Dawley rats following injection into the gastric submucosal layer was demonstrated. In addition, a long-term tracing in vivo (from 4 days to 3 months) indicated that the DIONs had good biosafety and biocompatibility according to an evaluation of the behavior and blood biochemistry of the rat and a histopathological examination of the important organs.