Coeliac disease is an inflammation of the small intestine triggered by gluten ingestion. We present a fluorescent genosensor, exploiting molecular-beacon-functionalized gold nanoparticles, for the identification of human leukocyte antigen (HLA) DQ2 gene, a key genetic factor in coeliac disease. Optimization of sensor performance was achieved by tuning the composition of the oligonucleotide monolayer immobilized on the gold nanoparticle and the molecular beacon design. Co-immobilization of the molecular beacon with a spacing oligonucleotide (thiolated ten-thymine oligonucleotide) in the presence of ten-adenine oligonucleotides resulted in a significant increase of the sensor response owing to improved spacing of the molecular beacons and extension of the distance from the nanoparticle surface, which renders them more available for recognition. Further increase in the response (approximately 40%) was shown to be achievable when the recognition sequence of the molecular beacon was incorporated in the stem. Improvement of the specificity of the molecular beacons was also achieved by the incorporation within their recognition sequence of a one-base mismatch. Finally, gold nanoparticles functionalized with two molecular beacons targeting the DQA1*05* and DQB1*02* alleles allowed the low-resolution typing of the DQ2 gene at the nanomolar level.