The diffusion of glucose and a series of poly(ethylene glycol)s (PEG, average molecular weight = 200, 600, 1000, 10000) in agarose gels (in the range of 0.5-3%, w/w) have been studied with a novel refractive index method developed in our laboratory. The change of the gel refractive index caused by the change of the diffusing solute concentration in the gel during the diffusion process enables the effective solute diffusion coefficients to be computed. The change in glucose concentration in agarose gel with the diffusion distance and the diffusion kinetics of glucose in agarose gels reveal that glucose diffuses freely within these gel matrixes with a high diffusion coefficient ∼6 × 10-10 m2/s for 0.5% and 1.5% agarose gels. However, the diffusion coefficients of PEGs in the gel are generally in the range of (0.7-5.9) × 10-10 m2/s and decrease with an increase in the network density (higher agarose gel concentration) and/or in the molecular weight of the diffusing solutes. It is observed that the molecular weight dependence of the PEG diffusion coefficients can be described with a power law expression with an exponent -0.533, indicating that PEG in agarose gel is in a situation close to the negligible solvent drainage coil (exhibited in dilute solution with a characteristic exponent -0.5). Furthermore, two models of both Ogston's model and Amsden's model based on the obstruction effect were used to simulate the effect of polymer volume fraction on the diffusion coefficients of the solutes in agarose gel to account for the results.