The traditional gold-nanoparticle-based lateral flow immunoassay (LFIA) cannot satisfy the requirements for the sensitive detection of dehydroepiandrosterone (DHEA) in human urine. To enhance the sensitivity of the LFIA, platinum-iridium nanocubes (Pt-Ir NCs) with high catalytic efficiency and stability were synthesized and labelled with polyclonal antibody (pAb) to form a pAb-Pt-Ir probe. For the detection of DHEA, a novel LFIA with Pt-Ir NCs as an optical label and an enhanced LFIA in which the peroxidase-like activity of the Pt-Ir NCs was triggered by the introduction of the chromogenic substrate 3-amino-9-ethyl-carbazole (AEC) were developed and compared with a LFIA with platinum nanocubes (PtNCs) as an optical label. The visual limit of detection was 0.5 ng mL-1 for Pt-Ir-LFIA and 0.05 ng mL-1 for AEC-enhanced Pt-Ir-LFIA, in comparison to 100 ng mL-1 for PtNCs-LFIA and 50 ng mL-1 for AEC-enhanced PtNCs-LFIA. The average recoveries from spiked urine samples ranged from 90.8% to 110.4%, with a coefficient of variation below 12.6%, suggesting the accuracy and reliability of our developed immunoassay. Achieving excellent sensitivity, specificity, and reproducibility, Pt-Ir-LFIA provided a promising platform for monitoring DHEA.