The development of unexpected red blood cell antibodies can significantly complicate transfusion therapy and result in more difficulties in cross-matching of blood. This study aimed to determine the occurrence rate of red blood cell alloimmunization in patients from Nanjing and the surrounding area. The antibody screening tests were carried out on 604 patients in Nanjing Red Cross Blood Center from January 2014 to December 2016, and the results were compiled and statistically analyzed. In the 604 patients, 483 cases revealed autoantibodies with or without underlying alloantibodies, while 121 patients had only alloantibodies in their serum. The overall frequency of alloimmunization was 32.5%. The most frequent antibodies were what against the Rh systerm(72.39%), followed by MN system (25.71%).