The invention discloses an O-carboxymethyl-N,N-double chain long alkylated chitooligosaccharide, and a preparation method and an application thereof. The chemical structure of the O-carboxymethyl-N,N-double chain long alkylated chitooligosaccharide is shown in the description. The preparation method comprises the following steps: protecting the amino group of chitooligosaccharide by adopting a micro-molecular aldehyde compound, carrying out a carboxymethylation reaction on the C6-position hydroxyl group of the amino group protected chitooligosaccharide by adopting chloroacetic acid, removing the protection group for protecting the amino group to obtain O-carboxymethyl chitooligosaccharide, and carrying out an alkylation reaction on two H in the amino group of the O-carboxymethyl chitooligosaccharide through adopting long-chain fatty aldehyde to obtain the O-carboxymethyl-N,N-double chain long alkylated chitooligosaccharide. The method makes the C6-position hydroxy group in the chitooligosaccharide be carboxymethylated and have hydrophobous long alkyl side chains, so the chitooligosaccharide is amphiphilic; and the prepared O-carboxymethyl-N,N-double chain long alkylated chitooligosaccharide has antibiosis and moisture retention property, and keeps the original good film forming ability, biocompatibility and biodegradability of the chitooligosaccharide.