A novel water-soluble cyclometallated iridium complex [Ir(pq-COOH)₂FDS]⁻ (pq-COOH = 2-phenylquinoline-4-carboxylic acid, FDS = 3-(2-pyridyl)-5,6-bis(4-sulfophenyl)-1,2,4-triazine dianions) (abbreviated as Ir) was synthesized and its phosphorescent property was comprehensively studied. It was found that the complex exhibited strong phosphorescence, which peaked at 634 nm in neutral conditions (maximized at pH 8.0). Its phosphorescence decreased with an increase in acidity of the aqueous solution. At pH 2.0, the quenched phosphorescence could be resumed upon the addition of human serum albumin (HSA) because of the hydrophobic and electrostatic interactions between HSA and Ir. Based on this phenomenon, a “turn on” type phosphorescence probe was developed for the detection of HSA. Under optimal conditions, a wide calibration range of 1–280 nM was obtained with a limit of detection of 0.8 nM for HSA. The phosphorescence probe was successfully used for the determination of HSA in blood serum and urine samples.