In this study, we developed the portable chemiluminescence (CL)-based lateral flow assay (LFA) platform for the detection of cortisol in human serum. Cortisol is well-known as a stress hormone due to its high relevancy for human mental and physical health, such as hypertension or depression. To date, a number of optical devices have provided the sensitive determination of levels of analytes. However, this modality type still requires costly optical modules. The developed CL platform is simply composed of two detection modules along with a loading part for the LFA strip. The LFA membrane contains gold nanoparticle probes conjugated with antibodies against cortisol and horseradish peroxidase (HRP), which can also efficiently increase the luminescent signal by providing many areas for anti-cortisol antibody and HRP. The measured voltage signals coming from the photodiode in a CL reader were compared with a standard microplate reader for the evaluation of accuracy. The linear range observed for cortisol was measured to be 0.78–12.5 μg/dL (R2 = 0.99) with a limit of detection (LOD) of 0.342 μg/dL. In addition, the CL-LFA reader showed a high correlation (R2 = 0.96) with the standard cortisol console (COBAS 8000, Roche), suggesting that our developed CL-based LFA platform can be usable in situ.