A universal nanochip for mass spectrometry analysis and preparing method and application of the same, relates to a technical field of mass spectrometry analysis. A main material of the nanochip is a silicon-based semiconductor material, array-type spotting wells are distributed at a surface of the main material, and an inner surface of the spotting well is of a nanostructure; the surface of the main material has a regional hydrophobic modification, and inside the array-type spotting well is a hydrophilic region and outside the spotting well is a hydrophobic region; or outside the array-type spotting well is a hydrophilic region and inside the spotting well is a hydrophobic region. The nanostructure can extract molecules on a surface of a biological tissue sample to be tested, and improves laser energy absorption and utilization, thereby improving ionization efficiency and enhancing mass spectrum signals. The universal nanochip can be widely applied to clinical inspection.