Langmuir-Blodgett (LB) films of an amphiphilic diacetylene (DA) were fabricated from the aqueous subphase where a water-soluble polymer, polyallylamine (PAA), was added by varying the concentrations. The effect of the PAA concentration on the structure, the morphology, and the polymerization in the DA/PAA LB film was investigated by measurements of XPS and IR spectra, atomic force microscopy (AFM), and the UV-visible absorption spectral changes on irradiation with UV light, respectively. The component ratio between DA and PAA and the ionic states of the components changed with the concentrations. The morphology and the polymerization behaviors depend strongly on the component ratio. At PAA concentration of 0.1 mM, nanofibers with a width of 30 Mm and a height of 2 nm were observed in the DA/PAA LB films. We have demonstrated that globular structures form the nanofiber structures.