The continued increase of global energy demand, depletion of fossil fuel, and deteriorated environmental conditions have triggered the need to search for potential sources of renewable energy. Biodiesel has been widely recognized to have promising potential. Nonetheless, it is critical to ensure the sustainability of biodiesel production to protect humans and conserve the environment, while achieving economic viability. From the sustainability point of view, it would be more effective to promote the sustainability feature in a process during the design stage rather than at the latter operational stage due to reduced engineering effort and cost. In this paper, a systematic framework for sustainability assessment of biodiesel production pathways during preliminary engineering stage is developed. In preliminary engineering stage, the major process modules (unit operations) with their main process streams are conceptualized in a process flow diagram (PFD). With the PFD, the data on mass and energy balance also become available to the users. With the input data from the PFD, the evaluation is based on the multiple objectives of inherent safety, health and environmental properties, as well as economic performance. Since multiple and conflicting objectives are involved, a fuzzy optimization approach is adapted into the framework. The overall framework is comprised of several major steps, i.e. process simulation, development of a mathematical optimization model, and production pathway optimization and ranking. To illustrate the proposed framework, an assessment is performed to identify the most optimal biodiesel production pathway among four pathways selected from a previous assessment study conducted during the research and development stage.