Cotton leaf spot (Alternaria macrospora) is a widespread disease that occurs in the main cotton-producing area of China. In managing this disease, a novel chitosan-based biopesticide, an amphiphilic N-alkyl-O-carboxymethyl chitosan derivative, was prepared. The product was selected from variations of chitosan with different molecular structures, which were obtained via a two-step reaction. First, carboxymethyl chitosans with varying molecular sizes were obtained by etherification with chloroacetic acid; then the carboxymethyl chitosan was alkylated with C4-C12 fatty aldehyde through a Schiff-base reaction. This procedure resulted in derivatives of amphiphilic N-alkyl-O-carboxymethyl chitosan, which showed strong antifungal activities against A. macrospora, and the efficacy was determined by its molecular structure.