Competitive lateral flow immunoassay (LFI) is a commonly used platform for detecting cortisol and other small molecules. However, its low sensitivity is a critical limitation for application in point-of-care tests. Thus, various signal enhancement methods have been developed, requiring complex operational steps and specialized detectors. In this study, we propose an automatic signal-enhanced LFI (asLFI), providing 100 times improved colorimetric sensitivity by applying 10,000-fold fewer AuNP conjugates compared to conventional competitive LFI, involving a single operational step. The incorporation of fewer AuNP conjugates improves the sensitivity of competitive LFI but does not produce a visible signal spontaneously. asLFI facilitates automatic signal enhancement without requiring additional operational steps, affording visible colorimetric signals. asLFI demonstrated high sensitivity in detecting cortisol (limit of detection = 3.8 pg mL