Interactions of 5,10,15,20-Tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TMPyP4) and 5,10,15,20-Tetrakis(N-propylpyridinium-4-yl)-21H, 23H-porphyrin (TPrPyP4) with the parallel four-stranded (TG4T)4 G-quadruplex DNA in 100 mM K+-containing buffer were studied using circular dichroism (CD) spectroscopy, visible absorption titration, and steady and time-resolved fluorescence spectroscopies. The results show that the binding stoichiometric ratios of both TMPyP4 and TPrPyP4 to (TG4T)4 are 3:1. Two types of independent and nonequivalent binding sites with the higher and lower binding affinities are confirmed, and the stronger and weaker binding constants are 9.44×107 and 6.94×105 M−1 for (TG4T)4-TMPyP4 complex, 7.86×107 and 6.35×105M−1 for (TG4T)4-TPrPyP4 complex, respectively. For both TMPyP4-(TG4T)4 and TPrPyP4–(TG4T)4 complexes, one porphyrin molecule stacks on the one end of G-quadruplex with the higher binding affinity, another two porphyrins bind weakly to the two external grooves. The size of cation side arms around porphyrin core almost fails to affect the binding mode, stoichiometry and affinity of porphyrin to (TG4T)4 G-quadruplex in 100 mM K+-containing buffer.