S-adenosyl-L-methionine (SAM) is one of most versatile molecules in nature and has wide medical applications. The ion-exchange separation process of SAM of the extract of yeast cells has many advantages over selective precipitation by picrolonic acid. Experiments of the dynamic column process of SAM on JK110 resin were carried out in a fixed-bed ion-exchange column. The effects of different operation parameters on the adsorption and desorption behaviors of SAM were investigated. The results show that the ion-exchange adsorption of SAM is successfully implemented at 2BV/h, 10 g/L, pH 5.0; the adsorbed SAM in the ion-exchanged bed is efficiently desorbed by 0.2 N H2SO4 solution at the flow rate of 2BV/h. According to material balance, the recovery yield of SAM for this ion-exchange process is 90.1%. Finally, this ion-exchange process was successfully scaled up to separate SAM at high yield and purity.