Layer-by-layer assembled microcapsules have the potential to be versatile cell delivery systems incorporating multiple activities and functions. However, it is necessary to determine the influence that different capsule locations have on activity of bioactive molecules in order to optimise delivery and for generation of multifunctional capsules. In this study we examine the influence that locating the bioluminescent enzyme luciferase in different microcapsule locations has on activity in intact synthetic and biodegradable microcapsules before and after cell delivery as well as its susceptibility to protease degradation. We also examine the effect of microcapsule position on cell transfection with plasmid DNA. Based on the findings of experiments in this study we also demonstrate co-delivery of luciferase protein and plasmid DNA encoding a fluorescent protein from two different locations within the same microcapsule. Our studies confirm that, the core, subouter layer, and outer layer are optimal for cell delivery but these positions offer least protection from protease activity. By contrast middle layer molecules remain entangled with capsule layers preventing their release which is inefficient for cell delivery but this provides better protection from protease degradation. The findings of this study will enable more rationale layer-by-layer assembly of microcapsules containing biologically active molecules for cell delivery and aid in the generation of multifunctional microcapsules.