Label-free electrochemiluminescence (ECL) immunoassays (lf-ECLIA), based on biomarker-induced ECL signal changes, have attracted increasing attention due to the simple, rapid, and low-cost detection of biomarkers without secondary antibodies and complicated labeling procedures. However, the interaction rule and mechanism between analytical interfaces and biomarkers have rarely been explored. Herein, the interactions between biomarkers and analytical interfaces constructed by assembly of a nanoluminophore and antibody-functionalized gold nanoparticles on an indium tin oxide electrode were studied. The nanoluminophore was synthesized by mixing Cu2+/l-cysteine chelate and N-(4-Aminobutyl)-N-ethylisoluminol-bifunctionalized gold nanoparticles with chitosan. It was found that positively charged biomarkers increased the ECL intensity, whereas negatively charged biomarkers decreased the ECL intensity. The assembly pH influenced the biomarker charges, which determined the ECL enhancement or inhibition. The detection pH only affected the ECL intensity but not the ECL changing trends. Based on the ECL signal changes, a charge-dependent lf-ECLIA was established, which exhibited inhibition responses to negatively charged human immunoglobulin G and copeptin and enhancement responses to positively charged cardiac troponin I, heart-type fatty acid binding protein, brain natriuretic peptide, and SARS-CoV-2 N protein. The linear range was 0.1-1000 pg/mL, and the detection limits were distributed in 0.024-0.091 pg/mL. Besides, a mechanism of the charge-dependent ECL enhancement and inhibition effects is proposed, which is very important for the development of new lf-ECLIA methodologies.