Abstract Bisphenol A (BPA)‐imprinted polymeric microspheres were synthesized by modified precipitation polymerization (MPP) method. Influences of cross‐linker, monomer, porogen volume, and agitation on polymerization were investigated. Proper amount of cross‐linker ethyleneglycol‐dimethacrylate (EGDMA) was critical to achieve narrowly dispersed microspheres. For template BPA, monomer 4‐vinylpyridine (4‐VP) was better than MAA to get the best imprinted effects. The optimum template/monomer ratio was 1:6. Increasing porogen volume increased size dispersity and decreased binding characters. Agitation increased coagulation and resulted in irregular particles. Microspheres with the best binding characters were used as selective stationary phase of chromatographic column to detect BPA in milk, pig urine, and chicken meat. Under optimal chromatographic conditions, the calibration graph was linear with R 2 = 0.9994 in the range of 3–50 μmol/L. The LOD and LOQ were 1 and 3 μmol/L, respectively. When large amounts (20 mL or 20 g) of samples were analyzed, the recoveries ranged from 70.2 to 87.3% with RSD less than 4.85% in all samples spiked with 0.05–0.2 μmol/L BPA. The intra‐day and inter‐day RSD were less than 1.83 and 3.96%, respectively. Microspheres prepared by MPP are successfully used in molecularly imprinted polymer (MIP)‐based analytical column to detect trace BPA in different biologic samples with acceptable accuracy and repeatability.