A label-free G-quadruplex-based luminescent switch-on assay has been developed for the selective detection of micromolar histidine in aqueous solution. In this study, an iridium(III) complex was employed as a G-quadruplex-specific luminescent probe while a guanine-rich oligonucleotide (Pu27, 5′-TG₄AG₃TG₄AG₃TG₄A₂G₂-3′)/cupric ion (Cu²⁺) ensemble was employed as a recognition unit for histidine. The initial luminescence of the iridium(III) complex in the presence of G-quadruplex DNA is effectively quenched by Cu²⁺ ions due to the Cu²⁺-mediated unfolding of the G-quadruplex motif. The addition of histidine sequesters Cu²⁺ ions from the ensemble, thereby restoring the luminescence of the system. The assay could detect down to 1 μM of histidine in aqueous media, and also exhibited good selectivity for histidine over other amino acids with the use of the cysteine, masking agent N-ethylmaleimide. Furthermore, the application of the assay for the detection of histidine in diluted urine samples was demonstrated.