Understanding the host-guest interactions in porous materials is of great importance in the field of separation science. Probing it at the single-molecule level uncovers the inter- and intraparticle inhomogeneity and establishes structure-property relationships for guiding the design of porous materials for better separation performance. In this work, we investigated the dynamics of host-guest interactions in core-shell mesoporous silica particles under in situ conditions by using a fluorogenic reaction-initiated single-molecule tracking (riSMT) approach. Taking advantage of the low fluorescence background, three-dimensional (3D) tracking of the dynamics of the molecules inside the mesoporous silica pore was achieved with high spatial precision. Compared to the commonly used two-dimensional (2D) tracking method, the 3D tracking results show that the diffusion coefficients of the molecules are three times larger on average. Using riSMT, we quantitatively analyzed the mass transfer of probe molecules in the mesoporous silica pore, including the fraction of adsorption versus diffusion, diffusion coefficients, and residence time. Large interparticle inhomogeneity was revealed and is expected to contribute to the peak broadening for separation application at the ensemble level. We further investigated the impact of electrostatic interaction on the mass transfer of molecules in the mesoporous silica pore and discovered that the primary effect is on the fraction rather than their diffusion rates of resorufin molecules undergoing diffusion.