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The lateral flow immunoassay (LFIA) is predominant in rapid diagnostic tests owing to its cost-effectiveness and operational simplicity. However, the conventional LFIA exhibits limited sensitivity and is susceptible to human variance for the result readout, impacting result interpretation. In this study, we introduced a novel one-step copper deposition-induced signal amplification lateral flow immunoassay (osa-LFIA) that markedly enhances the detection sensitivity for Staphylococcus aureus (protein A) and Pseudomonas aeruginosa (exotoxin A). Utilizing gold nanoparticles (AuNPs) as a catalyst, this approach employs ascorbic acid to reduce Cu2+ to Cu0, depositing on AuNPs at the test line and amplifying the signal. A user-friendly design features a three-dimensional paper structure incorporating pre-dried reagents, enabling a streamlined, efficient testing process. The osa-LFIA significantly lowers detection limits to 3 ng mL−1 for protein A and 10 ng mL−1 for exotoxin A, offering a tenfold improvement over conventional LFIA. Additionally, we developed a portable grayscale detection device, achieving less than 10% error in quantitative analysis compared to the data acquired and analyzed in the lab. This entire process, from detection to signal amplification, is completed in just 20 min. For the clinical trial, we utilized the osa-LFIA to test synovial fluid samples infected with Staphylococcus aureus. We also successfully detected different concentrations of the exotoxin A in parallel, with a recovery value of 96%–110%. Our findings demonstrate the osa-LFIA's potential as a rapid, highly sensitive, and simple-to-use diagnostic tool for detecting various pathogens, significantly advancing the field of rapid diagnostic testing. [ABSTRACT FROM AUTHOR]