The invention discloses a nano chip for mass spectrum detection. SiO2 particles are arranged at a bottom layer of the nano chip, wherein the particle size of the SiO2 particles is uniform and is not larger than 1 micron, the SiO2 particles have smooth surfaces; the nano chip has a rough metallic surface layer comprising gold, silver, platinum, palladium, aluminum, ferrum, titanium, copper, zinc, nickel, chromium, molybdenum, tin and barium. The invention also discloses a preparation method of the nano chip and application of the nano chip in mass spectrometry of a biological sample. The nano chip has better salt tolerance, and is applicable for quick, parallel and high-efficiency detection on practical biological samples. The nano chip disclosed by the invention has the advantages of simple preparation method, short preparation time, low economic cost and capability of being produced in batch, and therefore has a better application prospect and is worth of popularization and application.