To obtain the non-covalent molecular imprinted polymer with good adsorption properties, the interaction between the template molecule and functional monomer is importance. Molecular dynamics simulations and ultraviolet spectrum (UV) analysis were made to predict the interactions of the chloramphenicol (CAP) and different functional monomer (methacrylic acid (MAA), acrylicacid (AA), acrylamide (AM), methyl methacrylate (MMA)) complex. Firstly, the binding energy (ΔE) of CAP with different monomers is calculated by using the molecular dynamics method, and the ΔE follows the order of MAA>AA>AM>MMA. Secondly, the interaction strength between CAP and different monomers is studied by ultraviolet spectrum analysis, which is in the same order. Thirdly, the static adsorption experiments of the MIPMs and Non-Imprinted Polymers Microspheres (NIPMs) synthesized by using MAA, AA, AM or MMA respectively are studied, the results show that the adsorption performance of MIPMs for template are obviously better than the corresponding NIPMs, and the saturation adsorption capacity for different functional monomers have significant differences as well, which follows the order: MAA>AA>AM>MMA. Based on the experimental results, molecular mechanics calculation and UV analysis, it indicts that MAA is the most suitable functional monomer for CAP among the four monomers, and the molecular dynamics calculation and UV analysis can be used as effective tools for selecting functional monomer for the synthesis of MIPMs. Finally, molecular imprinted polymer microspheres (MIPMs) were prepared by using MAA as functional monomer. Scanning electron microscopy (SEM) shows that the obtained MIPMs has good dispersion, the average size was 8.44 μm，dispersion coefficient was 14.2%. The selectivity of MIPMs prepared by different molar ratio between CAP and MAA were studied by static adsorption method. The experimental results show that the static adsorption distribution coefficient of CAP is much higher than that of other analogs, has the obvious specific identification. In the CAP:MAA=1:4, the separation factor between CAP and Thiamphenicol (TAP) is as high as 4.57, higher than any other separation factor, this experimental result is consistent with the UV analysis results.