Background The majority of attenuated total reflection Fourier transform infrared (ATR FT-IR) investigations of cotton are focused on the fiber tissue for biological mechanisms and understanding of fiber development and maturity, but rarely on other cotton biomass components. This work examined in detail the ATR FT-IR spectral features of various cotton tissues/organs at reproductive and maturation stages, analyzed and discussed their biological implications. Results The ATR FT-IR spectra of these tissues/organs were analyzed and compared with the focus on the lower wavenumber fingerprinting range. Six outstanding FT-IR bands at 1 730, 1 620, 1 525, 1 235, 1 050 and 895 cm−1 represented the major C=O stretching, protein Amide I, Amide II, the O–H/N–H deformation, the total C–O–C stretching and the β-glycosidic linkage in celluloses, respectively, and impacted differently between these organs with the two growth stages. Furthermore, the band intensity at 1 620, 1 525, 1 235, and 1 050 cm−1 were exclusively and significantly correlated to the levels of protein (Amide I bond), protein (Amide II bond), cellulose, and hemicellulose, respectively, whereas the band at 1 730 cm−1 was negatively correlated with ash content. Conclusions The resulting observations indicated the capability of ATR FT-IR spectroscopy for monitoring changes, transportation, and accumulation of the major chemical components in these tissues over the cotton growth period. In other words, this spectral technology could be an effective tool for physiological, biochemical, and morphological research related to cotton biology and development.