The development of a piezoelectric biosensor based on nucleic acids interaction is presented focusing on the methodology for probe immobilization. This is a key step in any DNA biosensor development. Often, the detection limits and, in general, the analytical performances of the biosensor can be improved by optimizing the immobilization of the receptor on the transducer surface. DNA must be attached to the solid support, retaining native conformation, and binding activity. This attachment must be stable over the course of a binding assay and, in addition, sufficient binding sites must be presented to the solution phase to interact with the analyte. In this paper, the optimization of the coating of the gold quartz crystal surface, to immobilize an oligonucleotide probe, is reported. Two immobilization procedures are illustrated in details with a comparison regarding the immobilization of the probe, the detection of the hybridization reaction, and the possibility of regeneration. The two procedures are based on the use of biotinylated or thiolated DNA probes. Specific applications will be also presented.