DNA G-quadruplex (G4) could adopt multiple conformations, and the exact conformation is related to the presence of cations. However, the fact that cations with various concentrations could competitively bind with G4 is rarely investigated, which greatly limits the potential applications of G4-based sensors. Here, with PW17 (a G4-forming DNA sequence) as an example, Na+ and K+ with different concentrations competitively binding with PW17 are clarified by circular dichroism spectroscopy and electrospray ionization mass spectroscopy. Although Na+ could induce PW17 switching to unstable and antiparallel Na+-stabilized PW17 (2Na(+) -PW17) (C-Na(+) = 5-70 mM) and further to stable and hybrid 2Na(+)-PW17 (C-Na(+) = 70-800 mM), (C K+ = 0.1-10 mM) could replace Na+ in 2Na(+)-PW17 with 2Na(+)-PW17 transforming into Kt-stabilized PW17 (21(divided by-PW17). Moreover, the replacing ability strictly relied on C-K(+) and C-Na(+). In the switching process, a stable intermediate including a K+ and an Nat in one G4 (K+- Na+-PW17) is firstly detected. Importantly, the stable K+-Na+-PW17 is detected at 0.5 mM K+ and 140 mM Na+. Based on the facts, the interferences of Nat with the performance of PW17-based K+ sensors are investigated. With the stable K+-Na+-PW17 as a sensing probe and protoporphyrin IX (PPIX) as a G4 fluorescent read-out probe, a linear relationship between C-K( )+(500 nM-10 mM) and PPIX fluorescence is observed, which provides a fluorescence assay for detecting Kt with the co-existing 140 mM Nat This study exhibits clear evidence of Nat and K+ competitively binding with G4 and finds a novel and stable K+-Na+ PW17, which provides a clue to further explore G4 functions in Nat-contained samples.