Proteases are closely associated with many pathological conditions. Efficient detection of protease activity may be useful for diagnosis, prognosis, and the development of new therapeutic biomolecules. Fluorescent Resonance Energy Transfer (FRET) is defined as the non-radioactive energy transfer that occurs between two fluorophores. Fluorescent proteins are widely used in FRET biosensors because they can be genetically encoded and compatible with cells. Fluorescent Protein based FRET (FP-FRET) biosensors are used to monitor biological processes such as enzyme activity, intracellular ion concentration, conformational changes, protein-protein interactions. In this study, it was aimed to detect protease activity using an FP-FRET biosensor and TEV protease was chosen as a model enzyme. The plasmid encoding the mNeonGreen-TEV-mRuby3 fluorescent protein-based FRET biosensor was constructed. The gene of the designed FP-FRET biosensor was expressed in Escherichia coli DH5α cells using recombinant DNA techniques and purified using Ni-NTA affinity chromatography. As a result, the activity of the TEV protease enzyme was determined by emission measurements performed in the spectrofluorometer using the produced FP-FRET biosensor. The usability of the designed FP-FRET biosensor in the determination of protease enzyme activity was demonstrated.