The invention discloses a preparation method of konjac glucomannan gel microspheres. The preparation method includes: (1), adding konjac glucomannan into a water solution containing certain alcohol, and stirring to obtain a dispersion; (2), adding the dispersion into an oil phase, adding an emulsifier and alkaline substance, and stirring to obtain an emulsion; (3), adding an acidic solution and glutaraldehyde into the emulsion, and stirring for reaction to obtain gel microspheres; (4), using cyclohexane, alcohol and purified water to wash the gel microspheres to be neutral, and lyophilizing to obtain the konjac glucomannan gel microspheres. By the method, the konjac glucomannan gel microspheres which are uniform in grain size can be prepared and have porous structure. The method can be used for preparing media used for biochemical fine separation and gel microspheres serving as a drug conveying carrier, is expected to be capable of maintaining bioactivity and biostability of drugs and can be used for essence adsorption and controlled-release carriers. Due to small grain size, the konjac glucomannan gel microspheres can be dispersed in media well and are free of settling after being placed for a long time.