Abstract Cordycepin is an antibiotic with excellent biological and pharmacological activities. In this work, surface imprinting technology was used for cordycepin purification. Gaussian 09 simulation software was used to screen out methacrylic acid and acrylamide as the functional monomers of molecularly imprinted polymers (MIPs). The synthesis was accomplished with modified silica gel as a matrix, cordycepin as a template, methacrylic acid and acrylamide as functional monomers, ethyleneglycol dimethacrylate as a crosslinking agent and azodiisobutyronitrile as an initiator. MIPs for cordycepin separation were characterized by scanning electron microscope, Fourier transform infrared spectroscopy, elemental analysis and the pore size distribution analysis method. Adsorption isotherms for cordycepin show that the adsorption capacity using an initial cordycepin concentration of 16 mg/mL was 91.78 mg/g. According to the adsorption kinetic curve, MIPs obtained the maximum adsorption amount at 95.37 mg/g. In the adsorption kinetic experiments, adsorption reached equilibrium at 14 h with a rapid increase up to 4–6 h. The selectivity coefficients of MIPs were 3.14 (cordycepin versus inosine) and 3.74 (cordycepin versus adenosine). Experimental results indicated that the adsorption capacity and selectivity of MIPs were sufficient for the separation of cordycepin. MIPs were used to separate cordycepin from Cordyceps militaris . Cordycepin with 98% purity was obtained with the recovery of 25.67%. This result showed that MIPs prepared in this method have a good application prospect in the separation of cordycepin.