AbstractTriplet-triplet annihilation upconversion nanoparticles have attracted considerable interest due to their promises in organic chemistry, solar energy harvesting and several biological applications. However, triplet-triplet annihilation upconversion in aqueous solutions is challenging due to sensitivity to oxygen, hindering its biological applications under ambient atmosphere. Herein, we report a simple enzymatic strategy to overcome oxygen-induced triplet-triplet annihilation upconversion quenching. This strategy stems from a glucose oxidase catalyzed glucose oxidation reaction, which enables rapid oxygen depletion to turn on upconversion in the aqueous solution. Furthermore, self-standing upconversion biological sensors of such nanoparticles are developed to detect glucose and measure the activity of enzymes related to glucose metabolism in a highly specific, sensitive and background-free manner. This study not only overcomes the key roadblock for applications of triplet-triplet annihilation upconversion nanoparticles in aqueous solutions, it also establishes the proof-of-concept to develop triplet-triplet annihilation upconversion nanoparticles as background free self-standing biological sensors.