•Non-invasive diagnostic technique for β-thalassemia point mutations by SPR biosensor.•SPR biosensor based on novel hybridization format for point mutations identification.•Diagnostic approach reliable, reproducible and with non-invasive sampling. Optical biosensors based on Surface Plasmon Resonance (SPR), such as the Biacore™ X100, are widely used to study in real-time and in label-free mode bio-molecular interactions, including those allowing the identification of single point mutations responsible of genetic diseases, such as thalassemia and cystic fibrosis. The aim of this study was to verify whether the Biacore™ X100 can be proposed for the real-time detection of four mutations of the human β-globin gene causing β-thalassemia, a genetic blood disorder associated with absence (β0) or reduction (β+) of adult hemoglobin and severe anemia. In particular we analyzed the most frequent thalassemia point mutations present in the Mediterranean area (β039, β0IVSI-1, β+IVSI-110 and β+IVSI-6) using a novel SPR-based interaction format where two oligonucleotide probes (one complementary to the normal sequence and the other to the mutated one) were immobilized on sensor chips and asymmetric PCR targets obtained from genomic DNA of analyzed subjects were injected. For the development of the diagnostic approach, genomic DNAs of different genotypes for each mutation were obtained from blood samples or salivary swabs of 71 subjects, including healthy individuals, heterozygous β-thalassemia carriers and homozygous β-thalassemia patients. The results obtained allow proposing a new non-invasive diagnostic SPR-based protocol for thalassemia single point mutations using blood samples and salivary swabs as a source of genomic DNA.