The invention relates to a detection method of phosphorylation peptide, in particular to a detection and identification method for multi-phosphorylation site peptide segment in electrospray mass spectrum. The method includes the following steps that: the treatment of phosphorylation peptide is completed through adopting phosphatase; through controlling the dephosphorylation degree of phosphorylation peptide, treated sample and untreated sample are respectively analyzed and compared electrospray mass spectrum, thereby measuring phosphorylation peptide segment and determining the phosphorylation site of the segment. The invention has the advantages that: the method can be used to judge whether multi-phosphorylation peptide segment exists in electrospray mass spectrum; meanwhile, the invention can improve the mass spectrum response signal and detection rate of multi-phosphorylation peptide segment; moreover, through controlling reaction condition, the invention can speculate on the phosphorylation site of multi-phosphorylation peptide segment, and can also be used as a verification method in multi-stage mass spectrum analysis; in addition, the invention has simple method, convenient operation and controllable condition and easily realized online analysis.