An efficient sample clean-up and preconcentration procedure for phytoestrogens analysis in urine has been developed. It was based on a combination of solid phase extraction with hollow-fiber supported liquid membrane and molecularly imprinted beads (MIPs-HF-SLM-SPE). The molecularly imprinted polymers (MIPs) were synthesized by precipitation polymerization technique with biochanin A (BCA) as a template, giving narrowly dispersed microspheres with a regular shape. As the functional monomer, (dimethylamino)ethyl methacrylate (-DEM) turned out to be better than methacrylic acid (MAA) to get the best-imprinted effects. The MIPs used as sorbents in the MIPs-HF-SLM-SPE extraction process exhibited excellent binding selectivity for BCA, in comparison to non-imprinted polymers as well as its structural analogs (genistein and daidzein). Finally, the developed method was used to detect BCA in urine. Under optimal extraction conditions, the recovery of BCA in urine samples (using 4.5 mL sample spiked with 10 μg L-1) was over 41%, with a coefficient of variation (CV) < 6.6% (n = 5). The detection limit (LOD) and quantification limit (LOQ) for BCA analysis in urine were 0.41 and 1.36 μg L-1, respectively.