A preparation method of theaflavine belongs to agricultural product processing field, which relates to tea comprehensive processing. The invention resolves the problem of prior art as high cost in theaflavine preparation. The technical scheme comprises breaking fresh tea at low temperature, adding biological catalyst as plant leave juice, controlling the pH value of reaction liquid in the range of 4.0 to 6.0, feeding air at 25-50D EG C, mixing and reacting for 20-60min, separating solid and liquid after the reaction, feeding protecting agent into supernatant, concentrating at vacuum, directly atomizing, freezing and drying to obtain theaflavine product, or adding 95% alcohol of 1-3 powered volumes into supernatant, mixing for 20-40min, treating centrifugally to obtain supernatant and deposits, purifying the supernatant via macroporous adsorption resin, adding protecting agent, concentrating at vacuum, atomizing, freezing and drying to obtain theaflavine product. The preparation method of theaflavine confirms the stability of theaflavine and reduces production cost.