Herein, we developed a novel electrochemiluminescence (ECL) sensing interface based on Mg/Fe layered double hydroxides nanosheets (LDHs) for miRNA-135b detection in human colorectal cancer tissues. The LDHs nanosheet with large specific surface area and abundant active sites can work as the electrode sensing interface to effectively catalyze the electrochemical reactions. Furthermore, the formation of hydrogen bonds between 4-carboxyphenylboronic acid (CBPA) on the copper nanoclusters (Cu NCs) and fluorine ions limited the intramolecular vibration and accelerated electron transfer, greatly improving the luminescence signal. Due to the catalysis of LDH and the aggregation-induced enhanced electrochemiluminescence (AIECL) effect, the ECL quantum yield of Cu NCs has been significantly amplified. The Mg/Fe LDH-based sensing system for miRNA-135b detection had a linear detection range of 10 fM to 10 μM with a detection limit of 3.5 fM. Finally, the cancer tissue extracted from colorectal cancer patients have been successfully detected. The result indicated that the Mg/Fe LDHs sensing interface provided a new and reliable pathway for ECL analysis performance.