The invention relates to a method for identifying methylation sensitive sites of BstUI based on G-quadruplex, and belongs to the field of biochemical analysis. The method comprises the steps that first, Hairpin DNA containing a G-quadruplex sequence and modified by different methylation is used as a substrate, BstUI is added, reaction is performed at 60 DEG C, Hairpin DNA substrate containing themethylation sensitive sites of the BstUI cannot be recognized, and thus, the Hairpin DNA substrate containing the BstUI methylation sensitive sites cannot be cut, but Hairpin DNA substrate without themethylation sensitive sites of the BstUI can be cut to release G-quadruplex fragments; and finally, N-methylmesoporphyrin IX is added to the system and combined with the G-quadruplex to generate a strong fluorescent signal, and the methylation sensitive sites of the BstUI can be identified based on the strength of the fluorescent signal. In addition, DNA can further be characterized by gel electrophoresis to determine the methylation sensitive sites. The method provides two flexible identification methods and identifies new methylation sensitive sites of the BstUI.