Fourier transform infrared spectroscopy (FTIR)associated with horizontal attenuated total reflectance (HATR)was firstly used to diagnose β-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 β-thalassemia major patients and control: (1)Because of decreasing hemoglobin, the peak intensities are obviously lower in β-thalassemia group that is consistent with index from routine hemoglobin diagnosis. (2)In 1 750-1 500 cm-1 region, slight decrease at 1 652 cm-1(α-helix), 1 638 and 1 628 cm-1 bands but mild increase at 1 682 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 1 440, 1 453, 1 479 cm-1 bands arising from CH2/CH3 deformation vibration of phospholipids but increased intensities at 1 150 cm-1 band originating from C—O stretching vibration of carbohydrate and at 1 081 and 1 053 cm-1 bands that are attributed to 2,3-diphosphoglycerate (DPG)in β-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.