Here we describe the preparation and characterization of a polyrotaxane consisting of monodisperse alpha,omega-diaminopoly(ethylene glycol) (28 mer) and alpha-cyclodextrins (alpha-CDs). The monodisperse alpha,omega-diaminopoly(ethylene glycol) was prepared by stepwise synthesis starting from tetrakis(ethylene glycol) coupled with preparative gel permeation chromatography. The complex between alpha-CDs and alpha,omega-diaminopoly(ethylene glycol) was treated with 2,4-dinitrofluorobenzene, which is large enough to prevent dethreading. The product was characterized by gel permeation chromatography (GPC) and UV-vis, H-1 NMR, C-13 NMR, and 2D NOESY NMR spectroscopies. The H-1 NMR and C-13 NMR spectra show that the product consists of alpha-CDs, poly(ethylene glycol), and 2,4-dinitrophenyl groups. The 2D NOESY NMR spectra show that a poly(ethylene glycol) chain is included in cavities of alpha-CDs. The number of alpha-CDs in a polyrotaxane was determined from the UV-vis and H-1 NMR spectra and optical rotation, Twelve alpha-CDs were found to be captured in a monodisperse poly(ethylene glycol) chain.