The development of biotransformation must integrate upstream and downstream processes. Upstream bioprocessing will influence downstream bioprocessing. It is essential to consider this because downstream processes can constitute the highest cost in bioprocessing. This review comprehensively overviews the most critical aspects of upstream and downstream bioprocessing in enzymatic biocatalysis. The main upstream processes discussed are enzyme production, enzyme immobilization methodologies, solvent selection, and statistical optimization methodologies. The main downstream processes reviewed in this work are biocatalyst recovery and product separation and purification. The correct selection and combination of upstream and downstream methodologies will allow the development of a sustainable and highly productive system.