The measurement of pH is greatly significant in monitoring physiological and biochemical states. In this work, a novel micellar ratiometric fluorescent probe featuring sophisticated energy-transfer (ET) behaviors with p-nitrophenol (PNP) as the energy acceptor and a triplet-triplet annihilation upconversion (TTA-UC) system as the energy donor was designed. The pH-induced molecular configuration of PNP determined the process for the transfer of energy from TTA-UC to PNP. The introduction of the TTA-UC system enabled probe excitation under a long wavelength and afforded a ratiometric signal for pH detection with excellent reliability over diverse interfering factors. This TTA-UC/ET pH probe demonstrated a high sensitivity to hydronium below nanomolar concentrations and an excellent anti-interference ability in serum samples, which provided a novel significant strategy for rapid and accurate detection of blood pH in vitro.