Fluorescence resonance energy transfer (FRET) using biotinylated β-galactosidase (βGAL) as donor and Alexa Fluor 350 (AF350) labelled avidin as acceptor has been investigated by means of steady-state fluorescence and time-resolved fluorescence spectroscopy. The donors are readily paired with acceptors through the well-established binding affinity of biotin and avidin. The fluorescence energy transfer efficiency was determined by the donor fluorescence emission and lifetime changes in the presence and absence of acceptor. The theoretical energy transfer efficiency and theoretical average distance between donor and acceptor after non-covalent binding was calculated by taking the distribution of tryptophan residues in βGAL and avidin as well as the location of AF350 in avidin into account, which agree with the experimental data. It is shown how information of the location of acceptor can be obtained. Further, the fluorescence intensity image of AF350 on biotinylated βGAL coated quartz surface through UV FRET has been recorded using deep UV laser-based fluorescence lifetime microscopy. The results demonstrate that (a) deep UV laser-based fluorescence lifetime microscopy is a simple and useful method to study UV FRET of proteins using intrinsic fluorescence, (b) structural information even in complex multi-donor systems can be obtained and (c) FRET signals can be obtained to detect binding events using the native fluorescence of proteins as multi-donor systems.