Fourier transform infrared spectroscopy (FTIR) associated with horizontal attenuated total reflectance (HATR) was firstly used to diagnose beta-thalassemia patients. With excellent linearity (r = 0.997) and reproducibility (RSD < 4%), FTIR-HATR shows an order-of-magnitude increase in IR absorption bands over the single-path transmission FTIR Based on above, spectra from 37 patients' and 68 health samples indicated several observable differences in IR vibrational spectra of the Hb lysates between the beta-thalassemia major patients and control: (1)Because of decreasing hemoglobin, the peak intensities are obviously lower in beta-thalassemia group that is consistent with index from routine hemoglobin diagnosis. (2) In 1750-1500 cm(-1) region, slight decrease at 1652 cm(-1) (alpha-helix), 1638 and 1 628 cm(-1) bands but mild increase at 1682 cm(-1) all demonstrate structure changes by both Fourier self-deconvolution and second derivative spectra. (3) More importantly, difference spectra substantially demonstrate decreased intensities at 1440, 1453, 1479 cm(-1) bands arising from CH2/CH3 deformation vibration of phospholipids but increased intensities at 1150 cm(-1) band originating from C--O stretching vibration of carbohydrate and at 1081 and 1053 cm(-1) bands that are attributed to 2,3-diphosphoglycerate (DPG) in beta-thalassemia major group. Statistical analysis demonstrates significant difference of DPG/phospholipids ratio between two groups. All the samples can be 100% correctly classified into groups on the basis of this ratio. These finding could help understand possible mechanism for diagnosing thalassemia. It makes large-scale screening of thalassemia by FTIR a possibility.