Interferon-γ (IFN-γ) is a pivotal inflammatory cytokine whose abnormal expression is associated with autoimmune disorders, infectious diseases, and cancer. Herein, a signal-off electrochemiluminescence (ECL) immunosensor was developed by integrating catalytic signal amplification with oriented antibody immobilization. An amino-functionalized vertically ordered mesoporous silica film (NH2-VMSF) was rapidly grown on indium tin oxide (ITO) electrode by electrochemically assisted self-assembly (EASA), and gold-platinum bimetallic nanoparticles (AuPt NPs) were electrodeposited under nanoconfinement within its vertical nanochannels. The coupled Au and Pt sites promoted both dissolved oxygen (DO) reduction to superoxide radical anion (O2•−) and luminol oxidation, thereby enhancing luminol-DO ECL response. For construction of the biorecognition interface, glutaraldehyde was attached to the external film surface of NH2-VMSF while surfactant micelles still occupied the nanochannels, minimizing nanochannel blockage. Protein A was subsequently covalently coupled and used to orient anti-IFN-γ antibodies through their fragment crystallizable regions. IFN-γ binding formed an interfacial immunocomplex layer that hindered the transport of luminol, producing a concentration-dependent decrease in ECL intensity. The immunosensor can detect IFN-γ from 100 fg mL−1–100 ng mL−1 with limit of detection (LOD) of 31 fg mL−1. This platform provides sensitive strategy for cytokine analysis in complex biological matrices using catalytically amplified luminol-DO ECL system.