It is challenging to investigate fast supramolecular processes due to the lack of appropriate characterization methods with high structural resolution. In this study, microfluidic NMR spectroscopy as employed to monitor the kinetics of threading and dethreading of a pseudorotaxane system. By employing the high-time resolution of microfluidic NMR, 1H and 2D-ROESY NMR spectra were recorded at any time point 1.5 s after the onset of the process. By employing this technique, kinetic intermediates and rate-determining steps were successfully identified, which is not possible by other spectroscopic techniques. Our study demonstrates the capability of microfluidic NMR in investigating the mechanism of complex supramolecular systems.