AbstractHuman exposure to bisphenol compounds (BPs) has been implicated in the development of several chronic diseases. Instead of exploiting the traditional methods for determination of BPs, this work confirms that the human estrogen receptor α ligand binding domain (hERα‐LBD) is a powerful recognition element that can be used to monitor multi‐residue of BPs in urine samples by fluorescence polarization (FP) assay. Test parameters were optimized for the best performance. Under the optimal conditions, the IC50 values of BPs are in the range of 0.04–1.61 μg mL−1. Recovery experiments were then performed to assess the accuracy and precision of the established method. The results detected by FP assay show good agreements with that of liquid chromatography–tandem mass spectrometry method with a fit of R2 = 0.9372 and 0.9640 for BPE and BPAP, respectively. A computational methodology, ligand‐based pharmacophore model, was also employed to further explore the broad‐specific of tested compounds. It was found that the two hydrogen bond acceptor features and one hydrophobic aliphatic feature were essential for the corresponding cross‐reactivity results from the FP assay. All these results suggest that the established method can be successfully applied to monitor the occurrence of BPs in urine.