The invention relates to the technical field of biology, in particular to a PAP staining kit as well as a preparation method and a staining method thereof. The kit comprises a hematoxylin staining solution, an EA / OG staining solution, a buffer solution and a cleaning solution, wherein the hematoxylin staining solution comprises hematoxylin, sodium iodate, aluminum sulfate, sodium sulfite, ethanol, ethylene glycol, glycerol, acetic acid and water; the pH value of the hematoxylin staining solution is 2.0-3.0; the EA / OG staining solution comprises orange G, phosphotungstic acid, bright green SF light yellow, water-soluble eosin, sodium sulfite, ethanol, ethylene glycol, methanol, isopropanol, acetic acid and water; the pH value of the EA / OG staining solution is 5.0-6.0; and the pH value of the buffer solution is 6.8-7.2. The PAP staining solution is uniform in staining effect, clear in cell structure, bright in cell coloring, easy to clinically observe and diagnose, convenient to prepare, simple in staining step, stable in effect, clear in cell contrast and long in service life.