The effect of ionic strength of agarose solution and quenching temperature of the emulsion on the structure and mechanical strength of agarose-based chromatographic adsorbents was investigated. Solutions of agarose containing different amounts of NaCl were emulsified at elevated temperature in mineral oil using a high-shear mixer. The hot emulsion was quenched at different temperatures leading to the gelation of agarose and formation of soft particles. Analysis of Atomic Force Microscopy (AFM) images of particle surfaces shows that pore size of particles increases with ionic strength and/or high quenching temperature. Additionally it has been found that the compressive strength of particles measured by micromanipulation also increases with ionic strength of the emulsion and/or high quenching temperature but these two parameters have no significant effect on the resulting particle size and particle size distribution. Results from both characterization methods were compared with Sepharose 4B, a commercial agarose-based adsorbent. This is the first report examining the effect of ionic strength and cooling conditions on the microstructure of micron-sized agarose beads for bioseparation.