Parkinson's disease (PD) is characterized by progressive dopaminergic cell loss in the substantia nigra (SN) and elevated iron levels demonstrated by autopsy. Direct visualization of iron with live imaging techniques has not yet been successful. The aim of this study is to visualize and quantify the distribution of cellular iron using an intrinsic iron hyperspectral fluorescence signal. The 1-methyl-4-phenylpyridinium (MPP+)-induced cellular model of PD was established in SHSY5Y cells exposed to iron with ferric ammonium citrate (FAC, 100 μM). The hyperspectral fluorescence signal of iron was examined using a high-resolution dark-field optical microscope system with signal absorption for the visible/near infrared spectral range. The 6-h group showed heavy cellular iron deposition compared with the 1-h group. The cellular iron was dispersed in a small particulate form, whereas the extracellular iron was aggregated. In addition, iron particles were found to be concentrated on the cell membrane/edge of shrunken cells. The iron accumulation readily occurred in MPP+-induced cells, which is consistent with previous studies demonstrating elevated iron levels in the SN. This direct iron imaging could be applied to analyze the physiological role of iron, and its application might be expanded to various neurological disorders involving metals, such as copper, manganese, or zinc.