Novel polymer monolithic rods were prepared by in situ polymerization in a glass capillary and then coupled with high-performance liquid chromatography (HPLC) for the analysis of trimethoprim, sulfadiazine, and sulfamethoxazole. The rods showed homogeneity, uniform surface and micropores, good solvent-resistant ability, and excellent adsorptive ability onto antibacterial synergist compounds and sulfonamides. The extraction conditions were optimized, including extraction solvent, desorption solvent, extraction time, desorption time, and shaking frequency. A method of determining trimethoprim, sulfadiazine, and sulfamethoxazole through polymer monolithic rods microextraction coupled with HPLC was developed. The linear range was 20 μg/L to 200 μg/L, and the detection limits ranged within 10.6 μg/L to 15.5 μg/L. The proposed method was successfully applied to trimethoprim, sulfadiazine, and sulfamethoxazole analysis in spiked honey samples.