Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, diagnostic non-invasive prenatal methods have been developed. As far as Y-chromosome identification, this might be important for therapeutic intervention on sex-associated pathologies such as Duchenne muscular dystrophy, hemophilia and congenital adrenal hyperplasia. Surface plasmon resonance (SPR)-based biosensors might be useful for these studies, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. In this study BiacoreTM X100 has been applied to identify the Y-chromosome in cffDNA obtained from plasma samples of 26 pregnant women at different gestational ages, analysing the binding between SRY-PCR products and an SRY-specific probe immobilized on the sensor chip. The results suggest that there is a statistically significant difference between samples collected by pregnancies carrying male or female fetus. Moreover cffDNA, obtained at early gestational ages (6th-7th week) and not detectable by conventional quantitative real-time PCR, can be discriminated with high accuracy and reliability using SPR-based biosensors (Breveglieri G et. al, Prenatal Diagnosis 2016, 36, 353–361) and pre-amplification steps. In conclusion these results should be considered as the basis of future developments for more extensive diagnostic trials.