Herein, we proposed a versatile G-quadruplex (G4)-tetrahedral DNA framework (G4-TDF) nanostructure functionalized origami microfluidic paper-based device (μPADs) for fluorescence detection of K by lighting up thioflavin T (ThT). In this work, TDF provided robust structural support for G-rich sequence in well-defined orientation and spacing to ensure high recognition efficiency, enabling sensitive fluorescence sensing on origami μPAD. After introducing ThT, the G-rich sequences extended from TDF vertices formed a parallel G4 structure, showing weak fluorescence signal output. Upon the presence of target K, this parallel G4 structure transitioned to antiparallel G4 structure, leading to a significantly increase in fluorescence signal of ThT. Benefiting from the outstanding fluorescence enhancement characteristic of the G4 structure for ThT and excellent specificity of the G4 structure to K plus satisfactory recognition efficiency with the aid of TDF, this origami paper-based fluorescence sensing strategy exhibited an impressive detection limit as low as 0.2 mM with a wide range of 0.5-5.5 mM. This innovative G4-TDF fluorescence sensing was applied for the first time on μPAD, providing a simple, effective, and rapid method for K detection in human serum with significant potential for clinical diagnostics.