Melamine was employed as an analogue template to prepare a molecularly imprinted monolithic column which presents the ability of selective recognition to trimethoprim（TMP） because of the similarity between melamine molecule and substructure of trimethoprim.Methacrylic acid was applied as a functional monomer and ethylene glycol dimethacrylate as a cross linker during the in-situ polymerization process.Under the optimal conditions,the imprinted monolithic column showed a selective recognition to TMP when using methanol-water（70∶30,by volume） as mobile phase,but not to other structurally similar compounds such as folic acid and methotrexate,et al.Melamine would not disturb the determination of TMP.The MIP monolithic column was applied in the determination of TMP in sulfamethoxazole and human serum.The result indicated that,for sulfamethoxazole,the linear range of TMP was in the range of 5.05-101 mg/L（r=0.999 6）,and the recoveries at two spiked concentrations of 37.9 mg/L and 50.5 mg/L were 104% and 90%,respectively.For human serum sample,the linear range of TMP was in the range of 5.05-60.7 mg/L（r=0.999 6）,and the recoveries of TMP at two spiked concentrations of 25.3 mg/L and 50.5 mg/L were 95% and 97%,respectively.