A simultaneous quantitative analysis method for the thalassemia screening indicators mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and hemoglobin (Hb) was developed with Fourier transform infrared (FTIR) spectrometers and attenuated total reflection (ATR) combined with partial least squares (PLS). A total of 380 human peripheral blood samples were collected, which were composed of 180 positive samples and 200 negative samples according to the criteria of hematological indicator screening for thalassemia. One hundred fifty samples (64 negative, 86 positive) were randomly selected from all samples as the validation set, the remaining 230 samples (136 negative, 94 positive) were used as modeling samples; and then the modeling set was further subdivided into calibration set (68 negative, 47 positive, and 115 in total) and prediction set (68 negative, 47 positive, and 115 in total) for 200 times. Comparison of experimental results show that the prediction effect of PLS models in mid-infrared (MIR) fingerprint region (1 600～900 cm-1) was significantly better those of PLS models in the full scanning region (4 000～600 cm-1), and model complexity is significantly reduced. Based on PLS model in MIR fingerprint region, the optimal numbers of PLS factors for MCH, MCV and Hb were 10, 10 and 6, respectively, and the root mean square error (M_SEPAve) and the correlation coefficient (M_RP, Ave) of prediction in the modeling set were 2.19 pg, 0.902 for MCH, 5.13 fL, 0.898 for MCV and 8.0 g·L-1, 0.922 for Hb, respectively. The root mean square error (V_SEP) and the correlation coefficient (V_RP) of prediction in the validation set were 2.22 pg, 0.900 for MCH, 5.38 fL, 0.895 for MCV and 7.7 g·L-1, 0.929 for Hb, respectively. The sensitivity and specificity for thalassemia screening achieved 100.0% and 95.3%, respectively. Conclusion: FTIR/ATR spectroscopy combined with PLS method could provide a new reagent-free and rapid technique for thalassemia screening for large populations.