In vivo, brain imaging poses several challenges due to the complexity of brain tissue and the requirements for effective and safe imaging. In this manuscript, we reported on developing a new fluorescent dye, Cy5-PEG2, that can selectively accumulate in mitochondria, allowing us to visualize these organelles in different cell lines. Its versatility makes it broadly applicable in various experimental conditions, including monitoring mi-tochondrial dynamics in living cells. Moreover, we demonstrated that the Cy5-PEG2 dye could penetrate the blood-brain barrier (BBB), making it a promising tool for in vivo brain imaging. We further examined the re-sponse of glial cells in the hippocampus and neocortex regions of the brain through biomarkers GFAP and Iba1 but found no significant increase in their expression levels. This indicates that Cy5-PEG2 dye had no noticea-ble adverse effects on the brain's immune response or neuronal health. We anticipate such a mitochondria-targeting fluorescent dye can be used to facilitate the study of mitochondrial dynamics and function in the context of whole-brain physiology and disease progression. Further research into the safety and efficacy of this dye may be necessary.