<p>Residues of 17β-estradiol (E2) and diethylstilbestrol (DES) in animal-derived foods pose significant reproductive and carcinogenic health risks. This study presents an innovative electrochemiluminescence (ECL) biosensing platform for the ultrasensitive and sequential detection of E2 and DES. The sensor features a robust interface constructed by immobilizing Ru(bpy)₃²⁺-doped ZnOx-incorporated zinc oxalate metal-organic framework (Ru@ZnOx), electrodeposited gold nanoparticles (Au NPs), and a thiolated capture probe onto an electrode. Crucially, a dual-amplification strategy coupled with switchable dual channels is designed. A CHA-DNAzyme system specifically amplifies the E2 signal, while an Exo Ⅲ-assisted system amplifies the DES signal. Both systems generate ferrocene-labeled signal probes, which quench the ECL emission of Ru@ZnOx upon hybridization with P DNA (signal-off mode). After E2 detection, the S1 probe is efficiently removed via strand displacement, enabling subsequent DES detection on the same sensor. In direct detection mode, it achieves wide linear ranges from 10^− 6 to 10 ng mL^-1 for both E2 and DES, with remarkably low detection limits (LOD) (S/<i>N</i> = 3) of 0.32 pg mL^-1 for E2 and 0.56 pg mL^-1 for DES. In the sequential mode for DES, the linear range is 10^− 5 to 1 ng mL^-1 with an LOD of 6.8 pg mL^-1. The platform exhibits high stability, reproducibility, and specificity toward potential interferents, yielding excellent recoveries from 94.0% to 104.0% for determining E2 and DES in spiked milk samples. This work provides a highly reliable and sensitive method for monitoring trace levels of environmental estrogens, showcasing significant potential for ensuring the safety of animal-derived food products.</p> Graphical Abstract <p></p>