The invention discloses a DNA (Deoxyribonucleic Acid) bar code standard detection sequence of periophthalmus magnuspinnatus, application thereof and a molecular identification method based on the standard detection sequence, wherein the sequence of the DNA bar code standard detection sequence is shown by SEQ ID NO: 1. According to the application of the DNA bar code standard detection sequence of the periophthalmus magnuspinnatus, which is provided by the invention, the reliable DNA molecular identification technique adopted by individuals which are incomplete and cannot be accurately identified and a complete individual in specimens is realized; technical defects that the individuals which are incomplete cannot be basically identified and a professional person which is quite familiar with the morphology of the periophthalmus magnuspinnatus is needed for the identification of the complete individual, and the like, in the prior art are solved; in comparison with a conventional morphological identification method, the periophthalmus magnuspinnatus can be quickly and accurately identified.