Our previous studies have demonstrated that cotton fabrics treated with soluble immobilized-cellulase showed considerably lower degradation and higher retention of tensile strength than those treated with free cellulase. It is important to investigate the surface morphology and structure of cotton fibres for understanding the enzymatic degradation. In this study, the effects of the soluble immobilized-cellulase on the surface morphology and structure of cotton fibres were investigated. The ultrastructural changes in the fibre surfaces were inspected using Tapping-Mode Atomic Force Microscopy (TM-AFM) and scanning electron microscopy (SEM), and the results showed that the smooth surfaces could be obtained after the immobilized-cellulase treatment, and no obvious damage was observed on the fibre surfaces. The hydrogen bonds in the certain depth area beneath the fibre surface were investigated using Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy (ATR- FTIR ) after the cellulase treatments. Furthermore, the result of fibre accessibility indicated that the hydrolysis occurring in the interior of the cotton fibres was limited during the immobilized-cellulase treatment. Crystalline index (CI) of the cotton fibres treated with free cellulase was slightly higher than that of fibres treated with immobilized cellulase.