Bisphenol A (BPA) is an endocrine disrupter in environments which can induce abnormal differentiation of reproductive organs by interfering with the action of endogenous gonadal steroid hormones. In this work, the bisphenol A (BPA) molecularly-imprinted microspheres (MIMS) were prepared and used as biomimetic recognition material for in situ adsorption and selective chemiluminescence (CL) determination of BPA. Through non-covalent interaction, the BPA-MIMS was successfully prepared by Pickering emulsion polymerization using a BPA template, 4-vinylpyridine (4-VP) monomer, ethylene glycol dimethacrylate (EGDMA) cross-linker, and a SiO2 dispersion agent. The characterization of scanning electron microscopy (SEM) and energy-disperse spectroscopy (EDS) showed that the obtained MIMS possessed a regular spherical shape and narrow diameter distribution (25–30 μm). The binding experiment indicated BPA could be adsorbed in situ on the MIMS-packing cell with an apparent maximum amount Qmax of 677.3 μg g−1. Then BPA could be selectively detected by its sensitive inhibition effect on the CL reaction between luminol and periodate (KIO4), and the inhibition mechanism was discussed to reveal the CL reaction process. The CL intensity was linear to BPA concentrations in two ranges, respectively from 0.5 to 1.5 μg mL−1 with a detection limit of 8.0 ng mL−1 (3σ), and from 1.5 to 15 μg mL−1 with a limit of detection (LOD) of 80 ng mL−1 (3σ). The BPA-MIPMS showed excellent selectivity for BPA adsorption and the proposed CL method has been successfully applied to BPA determination in environmental water samples.