The invention proposes a preparation method of short-cut nitrification and denitrification granular sludge in high Do condition, which belongs to the technical field of waste water biological treatment. The short-cut nitrification and denitrification granular sludge mainly comprises bacilli and cocci and has higher nitrite depositing ability, denitrification ability and heterotroph activity so that the problem of free ammonia inhabitation caused when high NH4<+>-N selects and enriches nitrosation bacteria can be avoided and short-cut nitrification can be realized without increasing N2O release during short-cut nitrification in the high Do condition. The short-cut nitrification and denitrification granular sludge prepared by using the method has the advantages of compact granule structure and good stability, is suitable for treating the waste water with low COD (Chemical Oxygen Demand) and high NH4<+>-N, and can ensure the application of the short-cut nitrification and denitrification granular sludge in actual waste water treatment.