Thalassemia is a kind of blood diseases which has high morbidity and large influence. Previous methods for diagnosis are all very cumbersome and time consuming. By comparing Raman spectra of different kinds of thalassemia and normal erythrocytes at acid or alkaline pH, it was found that β-thalassemia and α-thalassemia erythrocytes have dissimilar Raman spectra in the acidic environment, such as the Raman spectra of β-thalassemia erythrocytes showed higher intensity at the characteristic bands assigned to oxyhemoglobin, and the characteristic bands assigned to deoxyhemoglobin were even completely replaced; β-thalassemia erythrocytes membrane has a smaller chain interaction between the transverse order parameters than normal erythrocytes, and the Slat values are different for different stages of anemia, while the Slat values are similar between α-thalassemia and normal erythrocytes, indicating that based on the effect of pH it is possible to diagnose thalassemia more quickly by using Raman spectra.