Developing a sensitive and reliable method for the screening of various endocrine-disrupting chemicals (EDCs) is in high demand and yet remains a significant challenge. Herein, we developed a CdSe/ZnS QDs-based nuclear receptor fluorescence probe (QDs-NRFP)-mediated biosensor for the screening of retinoic acid (RA)-active chemicals (a class of EDCs). The QDs-NRFP can be prepared on the spot via an antigen-antibody immunobinding interaction between the GST tag of the human retinoic acid receptor β ligand-binding domain (GST-hRARβ-LBD) and the CdSe/ZnS QDs-labeled anti-GST tag antibody. It can not only maintain the high binding activity of GST-hRARβ-LBD but also improve the sensitivity due to the high quantum yield of CdSe/ZnS QDs. Based on the indirect competition bioassay, the developed biosensor showed a detection limit of 1.8 ng/L all-trans-retinoic acid binding activity equivalent (atRA-BAE) with a linear range of 7.5-1183.6 ng/L. Compared with many cell-dependent in vitro assays, the QDs-NRFP-mediated biosensor is cell-free and unaffected by the cytotoxic substances in matrices and exhibited obvious superiority in detection time (within 40 min) and accuracy. As a case study, the biosensor was applied to detect RA binding activities in various sample matrices obtained from a wastewater treatment plant (WWTP) and physiological samples and showed satisfactory accuracy and reliability. The developed QDs-NRFP-mediated biosensor is expected to be capable of screening various EDCs with universality based on different nuclear receptor signaling pathways, which will substantially accelerate the assessment of global EDCs.