To improve knowledge on the biodegradation of cotton seed coat fragments in bioscouring, in this study Fourier-transform infrared (FT-IR) microspectroscopy was performed to analyze the composition of cotton seed coat. Microscope observation showed that cotton seed coat has a five-layer structure: epidermal layer, outer pigment layer, colorless layer, palisade layer, and inner pigment layer. Analysis of the FT-IR spectra from each layer shows that cutin, wax, cellulose, and pectin are the main components of the epidermal layer, while pectin and hemicellulose are the main constituents of the palisade layer, as well as aromatic and polyphenol, which are commonly considered as lignins. The main component of the outer and inner pigment layers is lignin. The results suggest that cellulase, pectinase, xylanase, and lignin oxidase are suitable for degradation of cotton seed coat. In addition, cutinase might be very promising in improving the enzymatic degradation of cotton seed coat.