The invention discloses a preparation method of large-particle-size agarose microspheres, which comprises the steps of providing an agarose solution having the mass concentration of 2-12% as a water phase; providing an organic solvent, in which an emulsifier is dissolved, as an oil phase; dropwise adding the water phase to the oil phase, and stirring to carry out emulsifying; cooling the emulsified mixed liquid for solidifying to obtain the large-particle-size agarose microspheres, wherein the agarose solution is prepared by dissolving at a temperature of 100-150 DEG C for 20-25 min, the emulsifier is one of, or a mixture of more than two of, span 40, span 60, span 80, span 85, tween 20, tween 60 and tween 80, the volume ratio of the water phase to the oil phase is 1:2 to 1:10, stirring speed is 50-1000 rpm, and emulsifying temperature is 50-100 DEG C. The invention also provides large-particle-size agarose microspheres prepared via the preparation method. The large-particle-size agarose microspheres have the particle size range of 400-1000 mu m, and have good pressure resistance, high temperature tolerance and high flow velocity resistance.