Herein, we have developed two enzyme-free magnetic relaxation assays for the detection of glucose utilizing the principle that glucose reduces Mn(IV) species to Mn(OH)2 in an alkaline environment, and the further conversion of Mn(OH)2 to Mn2+ under neutral condition can induce a noteworthy increase in the transverse relaxation rate (1/T2) of water protons. Thus, MnO2 nanosheets were first employed as probes and signaling units for the magnetic relaxation detection of glucose, achieving a detection limit of 1 μM. Furthermore, we realized straightforward magnetic relaxation detection of glucose by direct mixing of Mn2+ ions, NaOH and glucose in solution. This process encompasses the in-situ oxidation of Mn2+ ions to MnO(OH)2 and the subsequent reduction of MnO(OH)2 by glucose to Mn(OH)2, thereby integrating probe synthesis, and target identification within a one-pot reaction. To our knowledge, this type of analysis is more superior compared with the approach utilizing MnO2 nanosheets, since it exhibits higher sensitivity (with a limit of detection at 0.6 μM), faster, and enhanced convenience. Considering the advantages of being enzyme-free and facile, the magnetic relaxation sensing strategy, grounded in the redox reaction of Mn(IV) and glucose, holds substantial potential for diverse applications.