An efficient excitation energy transfer (EET) cassette, i.e.Ir-Np-OH, has been constructed by connecting an Ir(iii) complex phosphor as an acceptor to a naphthalimide fluorophore as a donor. Ir-Np-OH emits dual emissions, with one highly dependent on oxygen concentration and the other completely independent. Thus, Ir-Np-OH exhibits a sensitive and quantitative response to oxygen level in a ratiometric way. The linear response of Ir-Np-OH ranges from 0% (0 mmHg) to 10% (76 mmHg) oxygen tension, which matches very well with intracellular oxygen levels. And the ratiometric oxygen sensing of Ir-Np-OH is completely reversible. Also, importantly, Ir-Np-OH possesses a subcellular targetability specifically toward endoplasmic reticulum (ER), as proved by the co-localization imaging experiment. This feature of Ir-Np-OH should be noted, because generally speaking, it is much more challenging for small-molecule probes to target ER than other subcellular organelles, e.g. mitochondria. By using Ir-Np-OH, an apparent increase in oxygen level from 5.17% (39.29 mmHg) to 7.29% (55.40 mmHg) is monitored in ER of live MCF-7 cells, during the 30 minute stimulation with Rotenone, a typical inhibitor of the mitochondrial respiratory chain. In addition, upon ER stress directly induced by TG (Thapsigargin), the gradual increase in oxygen concentration is also clearly detected. These results confirm that Ir-Np-OH is a promising tool for oxygen-related cellular and chemical biology.