This study aimed to use micro-FTIR with transmission mode to investigate cellulose crystallinity of developing cotton fibers. Compared with ATR-FTIR method, we found that micro-FTIR can obtain more information of cellulose inside of the developing cotton fibers, especially in high wavenumber of 2800–3000 cm −1 region. Combined with curve fitting method, a new IR crystallinity index (CI) method named wax crystallinity index (WCI) was introduced to evaluate the cellulose crystallinity in the development of cotton fibers based on the peak and area ratios of 2900 cm −1 /2850 cm −1 and 2900 cm −1 /2920 cm −1 . The obtained WCI values demonstrated an excellent coefficient of determination with X-ray diffraction (XRD) CI method with the value up to 0.99. This study suggested that micro-FTIR was an effective technique to qualitatively analyze the crystallinity in developing cotton fibers combined with curve fitting method.