Codon optimization is a widely adopted strategy to improve heterologous expression in Saccharomyces cerevisiae, while other approaches, such as optimizing regulatory elements and cultivation conditions, are less common. Here, using enhanced green fluorescent protein (eGFP) as a heterologous reporter gene, we aimed to compare the effects of terminator selection, cultivation temperature, and coding sequence optimization in the S. cerevisiae strain 2805. Flow cytometry and immunoblotting were employed to assess changes in expression levels. While codon optimization still produced the largest improvement in expression, terminator choice and cultivation temperature also produced significant improvements in the expression of an already codon-optimized sequence. In particular, codon optimization was responsible for a 5-9-fold increase in the mean fluorescence intensity (MFI) compared with an increase of only 1.3-2.6-fold through terminator selection and an approximately 1.2-fold increase obtained by lowering the cultivation temperature from 30 oC to 20 oC. Among the 5 terminators tested, CPS1 produced the strongest effect on expression, increasing MFI by 2.6-fold compared with that obtained using the original Gal7 terminator. Taken together, the findings demonstrate that, within the tested strain and episomal expression system, terminator selection and reduced cultivation temperature can provide additional improvements after codon optimization and therefore serve as complementary steps in yeast expression optimization.