The invention belongs to the technical field of marine antifouling material preparation, and particularly relates to a preparation method of a microstructure surface with alternate hydrophilic-hydrophobic distribution. The method is characterized in that a monocrystalline silicon surface is covered by a photomask with a set pattern obtained through electron beam etching, and the set pattern with aset depth is obtained through etching; the etched monocrystalline silicon surface is used as a female die, and a hydrophobic organic silicon microstructure surface with a set structure is obtained through etching rollover; and through processes including plasma processing, hydrogen peroxide activation, hydrophobic liquid hole sealing, washing and hydrogel filling, an organic silicon microstructure is filled with hydrogel in an orderly manner, so that the regularly-structured microarray surface with the alternate hydrophilic-hydrophobic distribution is obtained. The method has the advantages that the height of the hydrogel in a microarray can be controlled to adjust projection area proportions and space area proportions of hydrophilic areas and hydrophobic areas in the microstructure surface with the alternate hydrophilic-hydrophobic distribution, so that a hydrophilic-hydrophobic microstructure is achieved, the broad-spectrum antifouling performance of a microstructure material is improved, and the method can be used for preventing and removing biofouling on the surfaces of ships and marine structures.