The purpose of this paper is to propose an estimation model to estimate water footprint of biofuel products which were produced from biomass crops as raw materials. In order to conserve natural resources and reduce carbon emissions, biomass energy was widely studied and developed. However, the increasing use of biomass energy also caused increasing demands of biomass crops and fresh water. Water footprint shows the water amount required to produce the biomass energy, and based on the literature the biomass crops provide more than 96% of the water footprint. In such a case, the operating water consumption is negligible in water footprint. This research found that the operating water footprint of second-generation bioethanol using rice straw and sugar cane bagasse as raw material was not negligible when the water footprint of biofuel products was less than a certain value. Therefore, this paper presents an empirical formula to estimate the water footprint of biofuel products using biomass crops as raw materials. This formula can help manufacturing engineers to estimate the water footprint in the planning stage, and then the selection decision of raw materials and manufacturing processes could be more thorough.