Luciferase is an enzyme that catalyses a reaction to produce a visible light using an oxidative process, a chemical reaction that is typically referred to as bioluminescent. Insects, bacterial origin or microorganism of marine nature were considered as the mainly sources of discovered luciferase. The protein was commercialized for biomedical and biotechnological use as reporter gene. The first discovered wild form of luciferase originally from Photinu spyralis (firefly). Hence, there is need for both exploration and examination of novel luciferase to be expanded to new sources such as fungal which may likely be exploited to serve commercial purposes and applications. In this study, a novel uncharacterized luciferase protein from a fungal species Verticillium longisporum, was modelled and analysed using bioinformatic tools. The modelled 3D structure is of high quality with a PROCHECK score of 99.5%, ERRAT2 value of 91.01%, and Verify3D score of91.01%, showing that the conformational structure is acceptable. The result showed that the fungal luciferase enzyme share major characteristics with luciferase representative from various fungal and bacterial species. There is only a slight difference in the two nucleotide bindings in V. longisporum with a D/E substitution of D with E and S/T substitution. The difference of the two nucleotides binding from the two proteins may be related to the evolutionary trends. Other differences include increased number of hydrophobic and polar amino acid groups than aromatic and aliphatic ones, as well as more coils and loops with less strands. The distance between the ligand and the binding site that houses Asp 64 and Thr 110 from template proteins (Riboflavin lyaseRcaE) and Asp 543 and Thr 589 from model luciferase is similar. The only difference occurred in the V. longisporum; protein oxidoreductase activities acts on paired donors, incorporate or reduce molecular oxygen, while in the template protein oxidoreductase activities act on single donors with incorporation of molecular oxygen. This study on fungal sourced luciferase present a unique opportunity away from the more well established bacterial and insect based luciferase.