Luminol is a well-known electrochemiluminescence (ECL) fluorophore that is applied in various sensing fields as an ECL reporter. Regulating the signal off/on transition of an ECL fluorophore offers great opportunities for sensors' design; however, such attempts on luminol are extremely scarce as it was regarded to lack promising modification sites. In this study, we developed four luminol derivatives with modification at the amine site and the enol site and systematically explored possible caging strategies to regulate ECL emission. The electrochemical and corresponding ECL properties reveal that the analogue with etherification on the enol group significantly inhibits the electrochemical oxidation behavior of luminol and almost entirely silences its ECL emission. Inspired by this observation, a new ECL probe luminol-O-β-d-glucose (Lum-Glc) was prepared by integrating glucose moieties and luminol through an ether bond. The ether linkage quenched the ECL emission of Lum-Glc, which could be cleaved by β-glucosidase (β-Glu), enabling the "turn-on" ECL determination of β-Glu. A broad linear detection range of 0.4-400 U/L with a low limit of detection of 0.1 U/L was achieved. This study provides new insight into mediating the ECL performance of luminol for the design of luminol-based ECL biosensors.