Singlet oxygen (O) is a widely used reactive oxygen species (ROS), making the development of new materials capable of efficiently generating it highly important. In this work, we report a series of isostructural metal-organic frameworks (MOFs), named JNU-300 (JNU = Jinan University), can spontaneously generate O by directly activating triplet oxygen (O) from the air without the need for additional light irradiation. The highly selective generation of O by JNU-300 is verified by electron paramagnetic resonance (EPR) spectroscopy and probe experiments for O. The generated O can be employed for aerobic oxidation of sulfide and antibacterial application. Mechanism studies revealed that the framework structure, and the adsorption and polarization of O by the coordination units of JNU-300, are crucial for the O generation. To the best of our knowledge, MOFs JNU-300 are the first MOF material capable of producing O by activating O in the absence of light at room temperature. This work provides new insights into the design and construction of functional MOF materials capable of generating O via activating O without light irradiation, opening new possibilities for their application in specialized fields that require O in the dark.