Detection of magnesium ion has been of great significance considering its critical physiological activities. Herein, we report ratiometric fluorescence detection of Mg2+ with high sensitivity and selectivity based on triplet-triplet annihilation (TTA) upconversion for the first time. Crown-ether functionalized anthracene derivatives were synthesized, which bifunctionally acted as not only annihilators to construct TTA upconversion systems but also the recognition probes for Mg2+ based on the photoinduced electron transfer (PET) mechanism. Their photophysical properties with the absence and presence of Mg2+ were comprehensively studied. It was found that solvents strongly influenced the photophysical properties and Mg2+-responsiveness. TTA upconversion systems with PtOEP as the sensitizer were further established and investigated. It turned out PtOEP/9-AEC in DCM exhibited an excellent linear relationship (R2 = 0.9979) between the intensity ratio (the integrated upconverted luminescence intensity (IUC) over the integrated downshifted phosphorescence intensity (IPL), IUC/IPL) and the concentration of Mg2+ under the excitation of 532 nm with a limit of detection value of 2.52 μM and a high selectivity to Mg2+. This work opened a new perspective of designs and applications of TTA-upconversion-based ratiometric fluorescence for ion detection.