The invention discloses preparation and application of a low-toxicity Kdo2-monophosphoryl lipid A containing five fatty acid chains, belonging to the field of bioengineering. By modifying Escherichia coli, a special-structure low-toxicity five-chain Kdo2-monophosphoryl lipid A is synthesized, and the Kdo2-lipid A molecule is not connected with the core polysaccharide long chain, and only composed of two 2-one-3-desoxyoctoic acids, five fatty acid chains and a C4' site single phosphoric acid. The Kdo2-lipid A is amphiphilic, is convenient for separation, extraction and purification, can be detected and directly identified, maintains the biological immunocompetence of the lipid A part, has obvious toxicity attenuation effect as compared with wild type W3110 LPS, and is a vaccine adjuvant with great development potential. Meanwhile, the method for extracting and purifying the Kdo2-lipid A is simple in steps and easy to operate.