The -3 fatty acid desaturase (3Des) is a key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs). However, the enzyme exhibits a significant preference towards different fatty acid substrates. To examine the molecular mechanism of its substrate specificity, a series of site-directed mutants were constructed based on the membrane topology model and functionally characterised by heterologous expression in Saccharomyces cerevisiae. Our results revealed that the W106F and V137T mutations markedly decreased the enzyme activity which indicated that these two residues were associated with substrate recognition. In contrast, the A44S, M156I and W291M mutations showed significant increments (30 to 40%) of the conversion rate for AA substrate desaturation, which suggests that these residues play a pivotal role in desaturation of longer chain-length substrates. Through homology modelling of 3-dimensional structures and molecular docking of FADS15, we propose that the critical residues that bind to the CoA groups may affect substrate localisation and govern substrate preference and chain-length specificity. Our work increases the understanding of the structure-function relationships of the microbial membrane-bound desaturases. The growing knowledge of the molecular mechanism will also aid in the efficient production of value-added fatty acids.