The invention discloses a preparation method for small-particle-size magnetic agarose microspheres. According to the preparation method, emulgator and hydrophobic organic solvent serve as an oil phase, mixed liquor of agarose and superparamagnetic Fe3O4 aqueous solutions serves as an aqueous phase, the aqueous phase is added into the oil phase under mechanical stirring to carry out pre-dispersion, then ultrasonication is carried out on pre-dispersion emulsion, and cooling, magnetic separation and purification are carried out on the crushed emulsion to obtain the small-particle-size magnetic agarose microspheres. The magnetic agarose microspheres prepared according to the preparation method have high magnetic response and superparamagnetism, and compared with commercially available products, the magnetic agarose microspheres have smaller particle sizes, larger specific surface areas, more active sites, and broad application prospects. Due to the fact that hydrophilic magnetic cores are adopted, through the modes of vortex vibration, ultrasonic dispersion, microwave heating, mechanical stirring pre-dispersion, ultrasonication carried out by a cell disruption instrument and the like, not only can the fact that the magnetic cores can be uniformly dispersed in the agarose solutions be guaranteed, but also the fact that the obtained magnetic agarose is small in particle size is guaranteed, and distribution is narrow.