The invention provides alpha-linolenic acid ester fat emulsion injection and a preparation method thereof. Alpha-linolenic acid esters, lecithin, glycerol, sterile purified water, an anti-oxidant, a stabilizing agent, a pH regulating agent and the like are taken as raw materials; and the preparation method comprises the following steps of: under the protection of nitrogen, mixing the raw materials, violently stirring, and shearing at a high speed to form primary emulsion; and emulsifying by using a high pressure homogenizing machine, filtering by using a microporous membrane filter, filling, sealing and sterilizing to obtain the alpha-linolenic acid ester fat emulsion injection. The alpha-linolenic acid esters are alpha-linolenic acid methyl ester, alpha-linolenic acid ethyl ester, alpha-linolenic acid ethylene ester, alpha-linolenic acid ethylene diester, alpha-linolenic acid propylene diester and alpha-linolenic acid triglyceride, and have the alpha-linolenic acid content of over 90 percent. The alpha-linolenic acid ester fat emulsion injection is applied in the fields of medical treatment and health, and can serve as nutritional and therapic medicine to be administrated through abenteric intravenous injection, intestinal tube feed or oral intake.