At present, there are no effective methods for the treatment of hemophilia B, and it has high mortality and disability. Therefore, it is very important for the carriers to carry out genetic counseling and make prenatal diagnosis. In this study, we made gene and prenatal diagnoses in a family with a novel F9 gene mutation, and report a novel F9 gene mutation.All exon sequences and flanking sequences of F9 gene were analyzed by Sanger sequencing in the proband; and then according to the F9 gene mutation in the proband, the F9 gene sequencing was performed on the family members. Based on the above results, the pathogenic mutation in F9 gene was finally identified, which was used for prenatal diagnosis.Sanger sequencing revealed c.1232G>C [p.Ser411Thr] mutation in F9 gene in the proband. c.1232G>C heterozygous mutation was also found in the proband's mother and grandmother, but male family members without hemophilia B had no this mutation. The analyses of amniotic fluid samples indicated positive sex-determining region on Y chromosome (SRY), and no c.1232G>C [p.Ser411Thr] mutation in F9 gene.We identified a pathogenic mutation in F9 gene in the family, made a prenatal diagnosis for the female carrier and reported a novel F9 gene mutation.