Iron is a crucial trace mineral vital for maintaining optimal human health and well-being. Despite being needed in small amounts, its importance cannot be overstated as it plays an important role in various physiological processes. In this research, we have developed a fluorescence probe utilizing starch functionalized with derivatives of rhodamine 6G to efficiently detect Fe3+ ions. This structure forms self-assembled starch/rhodamine 6G (RS0) fluorescent nanoparticles that exhibit strong fluorescence intensity under ultraviolet light. The ability of Fe3+ ions to increase the fluorescence of the RS0 probe enables the effective detection of Fe3+ ions. Importantly, this fluorescence increasing effect is specific to Fe3+ ions and remains unaffected by the presence of other metal ions. MTT assay against SH-SY5Y cells showed that RS0 was rather cytocompatible. Finally, SH-SY5Y cells were used to measure cell uptake and fluorescence imaging performances of RS0 which showed a good ability for live-cell fluorescence imaging.