The accurate knowledge of the diffusion behavior of protein within biomimetic hydrogel matrix at body temperature has a great implication for the design of efficient controlled release protein-base drug delivery devices. In this paper, we improved our previous in situ refractive index method with great temperature-controlled capability. For the first time, this newly improved method was employed to study the diffusion of protein (bovine serum albumin (BSA) and lysozyme) in agarose hydrogel at body temperature (37 degrees C). The change of the gel refractive index caused by the change of the diffusing protein concentration within the gel during the diffusion process enables the effective diffusion coefficients of protein to be estimated. The diffusion coefficients of proteins decrease with the increase of the concentration of agarose and the solute molecular size. At the considered range of agarose concentration (0.5-3.0 wt.%), the diffusion coefficients range from 4.98 to 8.21 x 10(-7) cm(2)/s for BSA and 1.15 to 1.56 x 10(-6) cm(2)/s for lysozyme, respectively. Temperature dependence of diffusivity of BSA in agarose hydrogel was also investigated. Furthermore, the retardance effect of polymer volume fraction on the diffusivity of both BSA and lysozyme in agarose hydrogels was analyzed with three models, Amsden's, Clauge and Philips', and Ogsten's model.