Immobilization of DNA is an important step in relation to DNA-based biosensors and bioassays with multiple applications. This unit describes synthesis and applications of novel bifunctional linker molecules containing nitrogen mustard and one of two types of functional groups: cyclic disulfide or biotin. Two ways of immobilizing DNA on a surface are described. With the first method, a bifunctional alkylating linker molecule is first reacted with the target DNA to form alkylated DNA and then immobilized on a specific surface. With the second method, the bifunctional alkylating linker molecule is first attached to the surface, and then the target DNA is immobilized through an alkylating reaction with a nitrogen mustard moiety. We have also achieved immunochemical detection and quantification of 5-methylcytosine in a target DNA immobilized by the above methods. The methods for immobilization of intact DNA using novel bifunctional linker molecules are applicable to a wide range of biological analysis techniques. © 2019 by John Wiley & Sons, Inc.