We present a method for preparing a quantum dot fluorescent monolayer film on a glass substrate. Since nanoparticles aggregate easily, it is difficult to prepare a nanoparticle monolayer film. We have used a covalent bond, the peptide bond, to fix quantum dots on the glass substrate. The surface of the quantum dot was functionalized with carboxyl groups, and the glass substrate was also functionalized with amino groups using a silane coupling agent. The carboxyl group can be strongly coupled to the amino group. We were able to successfully prepare a monolayer film of CdSe quantum dots on the glass substrate.