Objectives Hemophilia B (HB, OMIM: 300746) is one of the most common bleeding disorders with an X-linked recessive inheritance pattern, caused by the deficiency of coagulation factor IX (FIX). FIX is encoded by the F9 gene located on Xq27.1. Diagnosis of HB is primarily suspected by prolonged activated partial thromboplastin time (APTT), decreased FIX activity (FIX:C) or genetic test of the F9 gene. We herein described a Chinese family with patients of mild HB. Methods Sanger sequencing of the F9 gene was applied to identify mutation. Coagulation tests were performed. Results The proband was a 5-year-old boy. He suffered prolonged bleeding after tonsillectomy recently and circumcision last year as well. His grandfather experienced prolonged bleeding after gastric surgery. Both patients showed normal APTT, though they had significantly decreased FIX:C. Sanger sequencing of the F9 gene revealed a novel hemizygous F9 c.639C > A (p.Asn213Lys) missense mutation in both patients. The proband's mother carried heterozygous mutation. This mutation was located in the activation peptide domain of FIX. Conclusion In conclusion, we confirmed that APTT could be normal in mild HB patients. Highly sensitive APTT for mild HB and molecular genetic test could confirm the diagnosis of mild HB.