Highly selective detection of intracellular glutamine (Gln) is very essential to understand the roles of Gln in some biological processes. Here, we report a new fluorescent method for selective imaging of Gln in live cells with an aldehyde-containing iridium complex, [Ir(pba)2(DMSO)2]PF6 (Hpba = 4-(2-pyridiyl)benzaldehyde) (Ir1), as the probe. Density functional theory (DFT) calculation and experimental results suggest that the coordination and hydrogen bonding interaction between Ir1 and Gln synergistically stabilize the Ir1-Gln complex, modulate charge-transfer characteristics and emission of Ir1, and as a consequence, enable Ir1 as the probe for the fluorescent sensing of Gln. The sensing strategy is well-responsive to Gln without interference from other amino acids or Gln-containing peptides and is demonstrated to be useful for in situ Gln imaging in live cells. The study provides a new method for fluorescent imaging of Gln in live cells, which is envisioned to find interesting applications in understanding the roles of Gln in some physiological processes.