DNA G-quadruplexes (G4s) are believed to be involved in many biological processes, including the gene expression regulation and the maintenance of telomere. However, the kinetics of G4, in particular how G4 forms in the double-stranded genomic DNA is an underexplored issue. We found that a very stable G4 can form in the promoter of human myocyte enhancer factor 2D (MEF2D), the gene encodes a transcription factor belonging to the MEF2 (myocyte enhancer factor-2) family which regulates the response of heart to cardiac stress signals. Biophysical characterizations, in particular single molecule fluorescence resonance energy transfer (smFRET) measurements, suggest that this G4 is more stable than its duplex form in near physiological conditions. Two major G4 structures were identified based on single molecule conformational analysis on the end and internal labeled DNA systems. A free energy landscape for the G4 hybridization was established in light of the smFRET G4/duplex competition assay. In addition, the G4 hybridization and unfolding rate constants were obtained.