The dynamics of MinD protein has been recognized as playing an important role in the accurate positioning of the septum during cell division. In this work, spot tracking technique (STT) was applied to track the motion and quantitatively characterize the dynamic behavior of green fluorescent protein-labeled MinD (GFP-MinD) in an Escherichia coli system. We investigated MinD dynamics on the level of particle ensemble or cluster focusing on the position and motion of the maximum in the spatial distribution of MinD proteins. The main results are twofold: (i) a demonstration of how STT could be an acceptable tool for MinD dynamics studies; and (ii) quantitative findings with parametric and non-parametric analyses. Specifically, experimental data monitored from the dividing E. coli cells (typically 4.98 ± 0.75 µm in length) has demonstrated a fast oscillation of the MinD protein between the two poles, with an average period of 54.6 ± 8.6 s. Observations of the oscillating trajectory and velocity show a trapping or localized behavior of MinD around the polar zone, with average localization velocity of 0.29 ± 0.06 µm/s; and flight switching was observed at the pole-to-pole leading edge, with an average switching velocity of 2.95 ± 0.31 µm/s. Subdiffusive motion of MinD proteins at the polar zone was found and investigated with the dynamic exponent, α of 0.34 ± 0.16. To compare with the Gaussian-based analysis, non-parametric statistical analysis and noise consideration were also performed.