The main objective of this work was to gain novel information on the catalytic mechanism of Foot-and-Mouth Disease Virus 3C protease (FMDV 3Cpro) using ensemble and single-molecule fluorescence techniques. This was done to aid FMDV 3Cpro inhibitor development. FMDV is the causative agent of foot-and-mouth disease which affects cloven-hoofed animals, such as cattle and sheep, and can cause substantial losses to farmers as well as whole economies. Fluorescence anisotropy (FA) on ensemble level and fluorescence resonance energy transfer (FRET) on single-molecule level were used to probe the interactions of FMDV 3Cpro with its peptide substrates. The main focus of previous work has been on the substrate cleavage specificity of the 3Cpro. Therefore, the FA assays done here - using fluorescein-labelled and unlabelled peptide substrates - gave new insights into the substrate binding specificity of the enzyme. Single-molecule fluorescence experiments, in solution and with surface-immobilised enzyme, were done to probe the interaction of FMDV 3Cpro with FRET-labelled peptide substrates. It was demonstrated that single-molecule sensitivity could be reached, and that the peptide HiLyte488-APAKQLLC(HiLyte647)FDLLKK is a suitable FMDV 3Cpro substrate for single-molecule fluorescence experiments. Single-molecule TIRF (smTIRF) was used to look at the interaction of this FRET peptide with surface-immobilised FMDV 3Cpro, and it was shown that the enzyme retains its biological function after surface attachment. Furthermore, the smTIRF experiments yielded novel and detailed information on the catalytic mechanism of the protease, and the single-molecule data was shown to be consistent with existing ensemble results as well as ensemble fluorescence data obtained here. This work is the first demonstration of single-molecule level experiments on FMDV 3Cpro. A quick and convenient smTIRF protocol was developed, and the experiments done using this protocol yielded novel information on the mechanism of the enzyme. This information will undoubtedly be a step towards more rational FMDV 3Cpro inhibitor design.