Synthetic biology is a new research field which attempts to understand, modify, and create new biological entities by adopting a modular and systemic conception of the living organisms. The development of synthetic biology has generated a pluralism of different approaches, bringing together a set of heterogeneous practices and conceptualizations from various disciplines, which can lead to confusion within the synthetic biology community as well as with other biological disciplines. I present in this manuscript an epistemological analysis of synthetic biology in order to better define this new discipline in terms of objects of study and specific objectives. First, I present and analyze the principal research projects developed at the foundation of synthetic biology, in order to establish an overview of the practices in this new emerging discipline. Then, I analyze an important scientometric study on synthetic biology to complete this overview. Afterwards, considering this analysis, I suggest a three-level classification of the object of study for synthetic biology (which are different kinds of living entities that can be built in the laboratory), based on three successive criteria: structural hierarchy, structural origin, functional origin. Finally, I propose three successively linked objectives in which synthetic biology can contribute (where the achievement of one objective led to the development of the other): interdisciplinarity collaboration (between natural, artificial, and theoretical sciences), knowledge of natural living entities (past, present, future, and alternative), pragmatic definition of the concept of “living” (that can be used by biologists in different contexts). Considering this new theoretical framework, based on its potential objects and objectives, I take the position that synthetic biology has not only the potential to develop its own new approach (which includes methods, objects, and objectives), distinct from other subdisciplines in biology, but also the ability to develop new knowledge on living entities.