A reagent-free determination method for the thalassemia screening indicators hemoglobin (Hb), mean corpuscular Hb (MCH), and mean corpuscular volume (MCV) was developed based on Fourier transform infrared spectrometers equipped with an attenuated total reflection accessory. A random and stability-dependent rigorous process of calibration, prediction, and validation was conducted. Appropriate wavebands were selected using the improved moving window partial least squares method with stability and equivalence. The obtained optimal wavebands were 1722 to 1504 cm −1 for Hb, 1653 to 901 cm −1 for MCH, and 1562 to 964 cm −1 for MCV. A model set equivalent to the optimal model was proposed for each indicator; the public waveband of Hb equivalent wavebands was 1717 to 1510 cm −1 , and the public equivalent waveband for MCH and MCV was 1562 to 901 cm −1 . All selected wavebands were within the MIR fingerprint region and achieved high validation effects. The sensitivity and specificity were 100.0% and 96.9% for the optimal wavebands and 100.0% and 95.3% for the equivalent wavebands, respectively. Thus, the spectral prediction was highly accurate for determining negative and positive for thalassemia screening. This technique is rapid and simple in comparison with conventional methods and is a promising tool for thalassemia screening in large populations.