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Point-of-care testing using paper-based lateral flow assays (LFAs) has emerged as an attractive diagnostic platform. However, detecting small molecules such as cortisol using LFAs is challenging due to limited binding sites and weak signal generation. Here, we report the development of cortisol-specific aptamer-based probes and a paper-based origami preconcentrator (POP) to amplify the probe signal. The cortisol-specific aptamers were conjugated onto gold nanoparticles and hybridized with signal probes to create the cortisol-specific signal-on probe. POP, consisting of patterned layers with convergent wicking zones, induces electrokinetic preconcentration of the released signaling probes. By integrating cortisol-selective aptamer-based probes and POP, we accurately diagnosed cortisol levels within 30 min of signal probe incubation, followed by 10 min of preconcentration. Our sensor was able to detect cortisol levels in the range of 25–1000 ng/mL, with typical cortisol levels in plasma ranging from 40 to 250 ng/mL falling within this range. The successful detection of the wide range of cortisol samples using this approach highlights the potential of this platform as a point-of-care testing tool, particularly for lateral flow assay-based detection of small molecules like cortisol. Our approach offers a convenient and reliable method of cortisol level testing with a portable and accessible diagnosis device.