The invention relates to a high performance liquid chromatographic (HPLC) detecting method of the bioavailability content of nosipeptide preparation, which pertains to the biochemical technical field. Before the nosipeptide preparation is diluted, a mixing solution of an organic solvent and buffer solution is added for extraction; a high performance liquid chromatograph with ultraviolet-visible spectrophotometer is adopted, with a mobile phase with the ratio of acetonitrile: water phase equal to 50: 50, a flow velocity between 0.5ml/min to 2.0ml/min, and an anti-phase C18 macromolecular packing column, and a detecting wavelength between 230nm- 250nm. The areas of various secondary peaks are converted according to the area conversion coefficient (K), and the results are processed by the method for processing the main peaks, and the HPLC determination results are converted by the average conversion of content correction coefficient (P) to obtain the bioavailability content.