Herein, an electrochemiluminescence (ECL) biosensor was constructed for simultaneous and ultrasensitive detection of glutathione (GSH) and copper ion (Cu2+) due to thiol (captopril) stabilized gold nanoclusters (Au NCs) as a high-efficient ECL emitter. Impressively, we firstly demonstrated that thiol stabilized Au NCs (thiol-Au NCs) presented unexceptionable ECL performance over amino stabilized Au NCs (amino-Au NCs), due to fast electron transfer from massive Au-S electronic pathways in electrochemical excitation. Furthermore, GSH (target 1) and Cu2+ (target 2) could give rise to “on-off-on” ECL responses of Au NCs based on GSH-triggered competing reaction and Cu2+-specific-DNAzyme walker. As a result, the ECL biosensor achieved trace detection of GSH with a detection limit of 148 pM and Cu2+ with a detection limit of 91.5 fM. Thus, the strategy pointed out a definite direction to obtain ultra-bright ECL metal NCs for multiple biomarker testing, broadening the application in clinical diagnosis.