2-Hydroxy-3-isopropoxypropyl guar gum (HIPGG), which is a novel polysaccharide-based thermo-responsive polymer, was synthesized via grafting a hydrophobic reagent (isopropyl glycidyl ether (IPGE)) onto a hydrophilic main backbone (guar gum (GG)). The HIPGG exhibited reversible and tunable thermo-responsive properties. Changing the molar substitution (MS) of the hydrophobic side chain enabled the lower critical solution temperature (LCST) to be adjusted within the range of 29.6 °C to 43.7 °C. Fluorescence spectrometry, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were used to investigate the self-assembly behavior of HIPGG and the thermo-dependent size of its aggregates. It was demonstrated that HIPGG formed stable aggregates in aqueous solution, and the diameters of the aggregates increased as temperature increased. Subsequently, Nile red was used as a model to investigate the encapsulation and temperature-controlled release behaviors in HIPGG aggregates. The results indicated that Nile red was easily encapsulated in the hydrophobic region of HIPGG aggregates, and its release at 36 °C, 38 °C, and 42 °C revealed that temperature had a remarkable impact on release behavior.