A proliferation of chemical, reaction and enzyme databases, new computational methods and software tools for data-driven rational biosynthesis design have emerged in recent years. With the coming of the era of big data, particularly in the bio-medical field, data-driven rational biosynthesis design could potentially be useful to construct target-oriented chassis organisms. Engineering the complicated metabolic systems of chassis organisms to biosynthesize target molecules from inexpensive biomass is the main goal of cell factory design. The process of data-driven cell factory design could be divided into several parts: (1) target molecule selection, (2) metabolic reaction and pathway design, (3) prediction of novel enzymes based on protein domain and structure transformation of biosynthetic reactions, (4) construction of large-scale DNA for metabolic pathways, and (5) DNA assembly methods and visualization tools. The construction of a one-stop cell factory system could achieve automated design from the molecule level to the chassis level. In this article, we outline data-driven rational biosynthesis design steps and provide an overview of related tools in individual steps.