In the post-genome era, real-time monitoring of proteins is of great importance. The application of molecular beacons (MBs) for real time protein detection demonstrates great advantages in the understanding of many important biological processes involving two key biomolecules: nucleic acid and protein. In general, MBs are single-stranded oligonucleotide probes that have been designed to report the presence of complementary nucleic acids by fluorescence detection. MBs have also been used for protein recognition and protein/DNA interaction studies. Real-time monitoring of enzymatic reactions, such as the cleavage of single-stranded DNA by single-strand specific nucleases, has been effectively studied by MBs. The excellent signal transduction properties of MBs can be combined with the specificity of aptamers for protein binding for the development of a new generation of molecular probes, molecular beacon aptamer (MBA), for molecular recognition of proteins and for specific protein binding studies. Using this principle, new fluorescent probes have been developed for real-time protein monitoring, such as the MBA for ultrasensitive real-time monitoring of the platelet derived growth factor. These probes are highly selective and sensitive with detection limits in the sub-nanomolar range. MBs have also found unique applications in studying ligation and phosphorylation in real time and with excellent specificity.