The development of DNA probes started from 1950's for diagnostic purposes and it is still growing. DNA probes are applied in several fields such as food, medical, veterinary, environment and security, with the aim of prevention, diagnosis and treatment. The use of DNA probes permits microorganism identification, including pathogen detection, and their quantification when used in specific systems. Various techniques obtained success by the utilization of specific DNA probes, that allowed the obtainment of rapid and specific results. From PCR, qPCR and blotting techniques that were first used in well equipped laboratories to biosensors such as fiber optic, surface plasmon resonance (SPR), electrochemical, and quartz crystal microbalance (QCM) biosensors that use different transduction systems. This review describes i) the design and production of primers and probes, and their utilization from the traditional techniques to the new emerging techniques like biosensors used in current applications; ii) the possibility to use labelled-free probes and probes labelled with an enzyme/fluorophore, etc.; iii) the different sensitivity obtained by using specific systems; and iv) the advantage obtained by using biosensors.