Abstract: Objective: To establish the method for prenatal sex diagnosis of the fetal cells from maternal blood carrying sex-linked genetic disorder and to investigate the influence of several factors on ZFY automated cycle sequencing. Methods:The oligonucleotied primers designed with computer were located at Y chromosome specific ZFY（zinc-finger-Y） gene. The effects of DNA purity and DNA concentration, as well as the quality of water used during automated cycle sequencing were retrospectively analyzed. Results: When the concentration, purity and quality of water were not good, they would lead to difficulty in correctly judging the sequencing. Conclusions: The results indicated that fetal sex determination by nest PCR was practicable.And high purity,concentration of DNA and high quality of water should be used in automated cycle seqeencing.