The interactions between non-starch polysaccharides alter the physicochemical properties of the mucin, therefore can affect encapsulated nutrients penetrating through the mucus layer in the gastrointestinal tract. This study explored the interactions between mucin and galactomannans (GMs) with different mannose-to-galactose ratios going through contact, diffusion, and binding stages. During contact stage, the contact angle was used to characterize the interactions between mucin and GMs, and results showed that locust bean gum (4:1) with lower molecular weight had more contact area to form more hydrogen bonds with water. At diffusion and binding stage, based on the analysis of ATR-FTIR, rheology, and fluorescence spectroscopy, more galactose side chains in guar gum (2:1) had more hydrogen donors and acceptors and more space between polysaccharides to form intermolecular hydrogen bonds with mucin, which enhanced the interactions with mucin. Molecular docking results suggested that more hydrogen bonds in the main chain and less in the side chain resulted in more availability to form intramolecular hydrogen bonds and therefore reduced the strength of the interactions with mucin. Moreover, the Transwell assay revealed that enhancing the interactions of GMs with mucin, especially hydrogen bonds, slowed down the migration of the riboflavin. The current discovery laid the groundwork for designing GMs-based delivery systems that are capable to control the duration of nutrient action on the mucus by changing the molecular properties of the polysaccharide.