For signal amplification without an additional operation step in a gold nanoparticle (AuNP)-based lateral flow assay (LFA), a new and simple method utilizing two AuNP-antibody conjugates was developed. The 1st conjugate was the AuNP immobilized with an anti-troponin I antibody and blocked with bovine serum albumin (BSA), and the 2nd conjugate was the AuNP immobilized with an anti-BSA antibody and blocked with human serum albumin. The two conjugates were encapsulated in different pads, respectively. A scheme of the LFA system is described in the part A of first figure. The size of the two conjugates was very critical in the detection sensitivity of troponin I. When 10nm for the 1st and 40 nm for the 2nd were used, the detection sensitivity increased about a 100-fold compared to the conventional LFA. We could detect as low as 0.01 ng/mL troponin I in 10 min using the dual AuNP conjugate-based LFA, which was successfully applied in the analysis of serum samples of patients with myocardial infarction.