The invention discloses a preparation method of carboxymethyl chito-oligosaccharide. The method comprises the steps that 1, chitosan oligosaccharide is added into isopropanol, room temperature oscillationis conducted, andswellingchitosan oligosaccharide is obained; 2, a NaOH solution is added into the swellingchitosan oligosaccharide obtained in step 1; 3, an isopropanol solution of chloroacetic acid is added in alkaline feed liquid obtained in step 2, and a hydroxylation reaction is performed; 4, the feed liquid obtained after hydroxylation in step 3 is adjusted with an HCl solution to reachthe pH being neutral, then absolute ethyl alcohol is used for ethanol precipitation, and the feed liquid containing precipitation is obtained; 5, the feed liquidcontaining the precipitation in step 4is subjected to centrifugation, the precipitation is collected and washed multiple times, the washed precipitation is dried, powdering is conducted, and carboxymethyl chito-oligosaccharide powder is obtained. According to the preparation method, preparation is conducted through a chemical method, the carboxymethyl chito-oligosaccharide can be obtained, operation is easy, a high substitution degreeproduct can be obtained, and the product has the excellent antibacterial and antioxidant properties.