This study reports the fabrication of highly ordered rhombic gold nanocube superlattices (GNs) for surface-enhanced infrared absorption spectroscopy (SEIRAS) with significantly enhanced SEIRA effect. The SEIRA effect is controlled by manipulating the randomness of GNs, and finite difference time domain simulations reveal that the electromagnetic effect accounts for the improved spectroscopic vibrations. In situ SEIRAS results show that the vibrations of CO on Cu2O surfaces are enhanced by 2.4 ± 0.5 and 18.0 ± 1.3 times compared to traditional chemically deposited gold films in acidic and neutral electrolytes, respectively. Combined with isotopic labeling experiments, the reaction mechanisms for C-C coupling of CO electroreduction on Cu-based catalysts are revealed using GNs substrates. The study provides a novel approach with high sensitivity and reproducibility for revealing the reaction mechanisms of surface-mediated electrochemical reactions for renewable energy applications.This study reports the fabrication of highly ordered rhombic gold nanocube superlattices (GNs) for surface-enhanced infrared absorption spectroscopy (SEIRAS) with significantly enhanced SEIRA effect. The SEIRA effect is controlled by manipulating the randomness of GNs, and finite difference time domain simulations reveal that the electromagnetic effect accounts for the improved spectroscopic vibrations. In situ SEIRAS results show that the vibrations of CO on Cu2O surfaces are enhanced by 2.4 ± 0.5 and 18.0 ± 1.3 times compared to traditional chemically deposited gold films in acidic and neutral electrolytes, respectively. Combined with isotopic labeling experiments, the reaction mechanisms for C-C coupling of CO electroreduction on Cu-based catalysts are revealed using GNs substrates. The study provides a novel approach with high sensitivity and reproducibility for revealing the reaction mechanisms of surface-mediated electrochemical reactions for renewable energy applications.