The invention discloses a preparation method of low-molecular-weight and highly sulfated sea cucumber fucoidan. The preparation method comprises the following steps: by taking sea cucumber fucoidan as a raw material, firstly breaking a glycosidic bond by adopting an ultrasonic-microwave combined degradation method, decomposing the sea cucumber fucoidan to obtain low-molecular-weight sea cucumber fucoidan, then selecting methyl methanesulfonate and carrying out sulfation reaction with the low-molecular-weight sea cucumber fucoidan, so that the low-molecular-weight and highly sulfated sea cucumber fucoidan is obtained. The preparation method disclosed by the invention has the advantages that reaction cost is relatively low, preparation process is simple, reaction efficiency is higher, no pollution is produced, product structural damage is little, no side reaction is carried out, reaction is mild and controllable, and the obtained product is low in molecular weight and high in sulfate radical content, has stronger bioactivity, has the effects of resisting to oxidation, lowering blood glucose and reducing blood fat, is more beneficial to human absorption, can be applied to corresponding health product, functional food and medical formula industries and has extremely high social and economic significance and application potential.