The porous structure characteristics of twenty-one porous microspheres with agarose framework were investigated based on the low-field nuclear magnetic resonance (LF NMR) relaxation time (T (2)) distribution measurements. The feasibility of the technique was confirmed by direct relationship between T (2) and the mean pore size of the polymer networks (Rh) of agarose hydrogel which was established using the "Fiber-Cell" model. The expected pore radius distribution curve was obtained and the reliability was validated by comparing them with the results obtained by inverse size-exclusion chromatography. Thereafter, the technique was applied to characterize the pore size distribution (PSD) of soft microspheres and the pore size transformation of microspheres with or without grafted polymers in the process of protein adsorption. All of these results strongly support LF NMR as a promising, rapid and nondestructive technique for the determination of PSDs in many kinds of soft porous microspheres.