In this communication, we propose a new strategy and present the dual pH/O2 lifetime sensor based on covalent conjugation of fluorescein (pH sensor) and orthometalated iridium complex (O2 sensor) to human serum albumin (HSA). The resulting conjugate demonstrates biocompatibility, low toxicity, fast cellular uptake, and displays independent lifetime responses onto variations in media acidity and oxygen concentration that makes it suitable for application as effective pH/O2 probe in luminescent microscopy by using FLIM/PLIM detection mode. The concept applicability has been exemplified by dual spatially and temporally localized intracellular sensing of pH and O2 concentration in living cells.