A useful approach was proposed to easily synthesize molecularly imprinted adsorbent for the purification of tylosin from broth. Firstly, by molecular simulation based on density functional theory, methacrylic acid was chosen as a functional monomer by comparing the binding energy. Second, a novel method of polymerization based on precipitation polymerization with added seeds was used in water-mixed solvent for the preparation of water-compatible micron-sized MIP. Its static adsorption capacity for tylosin in aqueous solution was estimated to be 106.5 mg/g with the highest imprinting factor (IF) of 3.6. The selectivity coefficient (alpha) of tylosin to desmycosin was 3.3. The antibiotic in fermentation broth could be purified by means of molecularly imprinted solid phase extraction (MISPE), which allows MIP to be used for the purification of tylosin from a complex sample.