This study's purposes were to diagnose intractable hemolytic anemia and to provide guiding treatment for the affected family members. We performed NGS in a panel of 600 genes for blood diseases on a patient with obscure hemolytic anemia and her parents. We confirmed the diagnosis of pyruvate kinase deficiency, identified a novel homozygous mutation of the PKLR gene (NM_000298: exon 6: c.T941C: p.I314T), and ruled out other blood diseases in the Chinese family. Furthermore, amniotic fluid was taken from the mother during the second trimester, and DNA was extracted to analyze the type of PKLR gene mutation. The proband received cord blood and bone marrow from the second child of the mother for hematopoietic stem cell transplantation and achieved normal hematopoiesis. The genetic characterization analysis and genotype-phenotype correlation study of PKLR gene suggested that NGS was an effective method to confirm the molecular diagnosis of intractable hemolytic anemia. The identification of the mutation aided in prenatal diagnosis in the second pregnancy and the effective clinical management of the affected family.