Three-dimensional video imaging has emerged as an indispensable tool for real-time monitoring of dynamic acidic organelles. However, the limitation of video imaging is the absence of a specific stain for acidic organelle trackers. The aim of this work was to investigate the applicability of a potential acidic organelle tracker, Lyso-R, in three-dimensional video imaging in live cells. In a close examination of three differently designed rhodamine dyes, Lyso-R outperformed the other two with a suitable pKa value and higher membrane permeability. The uninterrupted fluorescence of Lyso-R towards macromolecules, e.g. lecithin and proteins, led to higher specificity and signal-to-background ratio than LysoTracker DND 189 and DND 99 for imaging acidic organelles. In addition, Lyso-R was photostable, and MTT assays confirmed its low toxicity towards cells. Inspired by these facts, three-dimensional tracking of a single acidic organelle in a live cell was obtained by staining with Lyso-R under confocal microscopy. The measurement of this organelle demonstrated that the distance change of the organelle centroid on XY plane was sharper than its depth change. The usage of Lyso-R was further extended with two-dimensional video imaging of acidic organelles during various cell metabolisms. All of these results demonstrate the potential applicability of Lyso-R as a three-dimensional imaging tracker of acidic organelles.