The investigation on the free motion of nanoparticles and their interaction with other media has become an attractive field for extending the practical application. However, the real-time monitoring of dynamic behaviors still exists significant challenge. In this paper, based on surface enhanced Raman spectroscopy (SERS) and the formation of “hot spots” during collision between Au nanoparticles and Au single crystal microplate under Brownian motion, the real-time monitoring of free motion behaviors of Au nanoparticles during collision and the dynamic SERS study were realized accordingly by using thiophenol (TP) as the probe molecule. The nature and influencing factors of microscopic motion of nanoparticles were investigated by statistical analysis of the “spikes” in the SERS trajectories, including “single spikes” and “cluster spikes”. The results reveal that the “spike” is mainly attributed to the “hot spots” formed by reversible collision of nanoparticle and plane. “Single spikes” correspond to the rapid departure of Au nanoparticles from the surface of the Au microplate after collision with the microplate, and “cluster spikes” correspond to the process of Au nanoparticles staying on the surface of the Au microplate for a short time after colliding with the plane and then leaving or possibly multiple nanoparticles colliding continuously. Increasing the concentration of nanoparticles is beneficial to the formation of “cluster spike”. The intensity distribution of the corresponding SERS characteristic peaks is concentrated in 5.2 cps and 8.7 cps, respectively. The relative intensities of SERS peaks of TP in the “spikes” are critically depended on the vibrational modes. It demonstrates that the probability of stretching vibrational modes is higher, and it is mainly due to the different orientations of molecules in the localized area during the dynamic collision processes. The realization of dynamic collision is beneficial to deeply understand the nature of microscopic motion of nanoparticles. It provides the basis for the investigation of dynamic interfacial chemical reactions in localized area.