A novel cyclometalated iridium(III) complex has been developed as a luminescent probe for human serum albumin (HSA) detection in aqueous solution. In this system, the initial luminescence of the iridium(III) complex in the absence of human serum albumin is weak. However, the addition of HSA could greatly enhance the luminescence intensity of iridium(III) complex. The assay could detect down to 0.05 μM of HSA, and also exhibited good selectivity for HSA over bovine serum albumin and other biomolecules such as ATP, β-amyloid, peroxidase, thrombin, lysozyme, trypsin, hemoglobin, and the various nucleic acids. Furthermore, the application of the assay for the detection of HSA in diluted urine samples and for monitoring the conformational changes of HSA induced by exogenous protein unfolding agents was also demonstrated.