This study examines the mobility of the restriction enzyme EcoRV along double-stranded DNA confined in nanofluidic channels, observed through fluorescence microscopy. Channels with diameters of 60–125 nm, equivalent to one to two times the DNA persistence length, enable precise investigation of DNA–protein interactions. The effects of base methylation on EcoRV movement are investigated in 60 nm channels, using DNA substrates in various methylated forms. Additionally, the role of DNA dynamics is explored in 125 nm channels by variation of DNA molecular weight. It is shown that EcoRV dissociates from DNA at both specific restriction sites and randomly between them, with dissociation at restriction sites occurring even when the enzyme is catalytically inactive. Base methylation increases binding affinity, suppresses dissociation, and prolongs EcoRV’s residence time in the bound (sliding) state, reducing overall mobility, a phenomenon similar to that observed for the nucleoid-associated protein Hfq. The presence of Mg2+ ions stabilizes the bound state, while ionic strength modulates EcoRV–DNA interactions through electrostatic screening. Furthermore, EcoRV’s diffusion behavior strongly correlates with DNA segmental dynamics, with slower dynamics associated with increased enzyme mobility. These findings suggest that DNA dynamics play a generic role in regulating protein mobility via transient binding, offering broader implications for understanding DNA search mechanisms and chromosome architecture.