Changes in the Synchrotron x-ray diffraction pattern of scalp hair were shown to occur in patients with breast cancer. A preliminary Fourier transform infrared (FT-IR) spectroscopy study of scalp hair using attenuated total reflection (ATR) supported the concept that these changes are due to an increase in the lipid content of the hair fibre. This study was undertaken to determine whether the ATR-FT-IR spectrum obtained using a single hair fibre ATR cell could be used in the detection of breast cancer. In addition, variable angle ATR-FT-IR difference spectra were obtained to investigate the location and the molecular structure of this lipid material in the hair fibre, which appears to be an indicator of breast cancer. Patients with breast cancer showed an increase in the peak height ratio of the 1446–1456 cm−1 C–H bending absorption bands of the ATR-FT-IR spectra of a single hair fibre. Peak height ratios > 1.0 were indicative of breast cancer. The spectra of scalp hair of subjects with breast cancer also showed a slight shift in C–H bending absorption from 1446 to 1448 cm−1 and from 1456 to 1458 cm−1 that could result from the formation of secondary structures by the increased lipid material. Variable angle difference spectra indicated that this increased lipid material is located in the cuticle–cortex interface area and appears to be similar to the lipids normally found here. An alteration in hair biosynthesis in the follicle caused by breast cancer signalling molecules, or biomarkers, is most likely involved. ATR-FT-IR spectral analysis of a long hair fibre containing a distal portion formed when the breast cancer was present and a proximal portion formed after the breast cancer was removed showed that hair fibre synthesis had become normal after the removal of the cancer. This study demonstrates the potential of ATR-FT-IR analysis of a hair fibre in the early detection of breast cancer and in studying how hair acts as a biosensor for breast cancer.