The hybrid promoter (hp4d) expression cassette, one of the efficient tools of Yarrowia lipolytica expression system, has been applied to produce or secrete a variety of recombinant proteins. This cassette directs a strong gene expression, because the hp4d promoter exhibits high level quasi-constitutive activity. The objective of this study is to test whether two expression cassettes inserted into a vector could function efficiently and simultaneously. Taking advantage of the well-known biosynthesis pathway of gamma-linolenic acid (GLA), we examined the performance of Y. lipolytica, transformed with two expression cassettes containing previously cloned Delta12-desaturase and Delta6-desaturase genes, by monitoring fatty acid composition of cellular lipids. Our results confirmed that each individual desaturase gene was expressed efficiently by the expression cassette. When two cassettes with respective desaturase genes, carried on the same vector, were integrated into yeast genome, a significant level of GLA was synthesized from endogenous linoleic acid (LA) and oleic acid (OA). Besides, both expression cassettes functioned effectively without influence from each other. These findings indicated that co-expression of two desaturase genes by this dual cassette vector was effective and simultaneous. Results from the present study provide an alternative approach for both the production of several proteins at the same time, and the development of single cell oil containing high-valued polyunsaturated fatty acids (PUFAs).