The invention discloses selenium-rich bio-fertilizer with a sustained release function and a preparation method thereof. The selenium-rich bio-fertilizer comprises, by weight, 280-300 parts of fertilizer body, biological microbial agents, 18-20 parts of functional carrier and auxiliary materials, wherein the biological microbial agents comprise, by weight, 0.08-0.09 part of Bacillus thuringiensis,0.1-0.15 part of Paenibacillus polymyxa, 0.2-0.25 part of Azotobacter chroococcum, 0.05-0.07 part of Bacillus subtitles, 0.1-0.15 part of Lactobacillus acidophilus, 0.1-0.15 part of Lactobacillus bulgaricus and 0.1-0.15 part of Streptococcus thermophilus; the functional carrier is prepared by mixing zeolite, charcoal, diatomite, sepiolite and bentonite according to the mass ratio of 3:1:1:2:1; the auxiliary materials comprise, by weight, 0.1-0.2 part of polyglutamic acid, 5-6 parts of potato powder, 0.1-0.2 part of humic acid, 5-6 parts of volvariella volvacea, 4-5 parts of garlic, 8-10 partsof pleurotus ostreatus, 4-5 parts of puffball, 5-6 parts of auricularia auricular and 12-13 parts of selenium ore. The selenium-rich bio-fertilizer and the preparation method thereof have the advantages that aiming at the feature that the absorption efficiency of vegetables or fruits to inorganic selenium is low, the selenium ore is ground into powder and prepared into a solution and a culture medium to culture fermentation fungi so as to obtain the fertilizer which can be well absorbed by plants, and the selenium content of the planted vegetables and fruits is increased.