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An AgNPs/N,S-GQDs paper-based electrode sensor for dual-potential detection of glutamate transaminase (GPT) activity was constructed by using synthesized AgNPs to modify the working electrode of the paper-based electrode and combining N,S-GQDs. According to the NADH produced by the GPT transamination reaction, the detection of NADH and GPT in two potential intervals of −1.7 V to 0 V and 0 V to 1.7 V, respectively, achieved good detection results. [Display omitted] • Construction of paper-based electrodes modified with AgNPs/N, S-GQDs. • Calibration of the profile of NADH produced as a result of the GPT transamination reaction. • Detection of NADH and GPT using AgNPs/N, S-GQDs paper-based electrodes at dual-potentials. • Detection principle based on transamination and electrochemical oxidation. The liver, one of the most vital organs in the human body, relies on glutamate pyruvate transaminase (GPT) as a sensitive indicator of hepatocellular injury, making it a cornerstone parameter in liver function assessment. This study employed a paper-based electrode modified with AgNPs/N, S-GQDs synthesized via pyrolysis to construct an AgNPs/N, S-GQDs paper-based electrode reaction system for detecting GPT activity across two potential ranges. This approach involved utilizing electrochemiluminescence (ECL) detection to indirectly measure GPT levels by detecting the NADH generated through GPT transamination, as well as investigating the relationship between GPT activity and the ECL intensity of the reaction system directly. Experimental results demonstrated a robust linear correlation between GPT activity and the ECL intensity of the reaction system within the ranges of 1U/L to 1000U/L and 5U/L to 200U/L, with detection limits of 0.24U/L and 3.73U/L, respectively. By establishing methods for indirect and direct GPT detection within the same reaction system across different scan ranges, this study enhances detection accuracy. The proposed method not only offers a wide linear range and low detection limits for GPT detection but also exhibits notable advantages in terms of detection efficiency compared to other methods, showcasing promising applications in the diagnosis and evaluation of liver function-related diseases. [ABSTRACT FROM AUTHOR]