This work presented a Spatially-Explicit-High-Resolution Life Cycle Assessment (SEHR-LCA) model for wastewater-based algal biofuel production, by integrating life cycle assessment, GIS analysis, and site-specific Wastewater Treatment Plants (WWTPs) data analysis. Wastewater resources, land availability, and meteorological variation were analyzed for algae cultivation. Three pathways, Microwave Pyrolysis, hydrothermal liquefaction, and lipid extraction were modeled for bio-oil conversion. This model enables the assessment of seasonal and site-specific variations in productivity and environmental impacts of wastewater-based algal bio-oil across the whole U.S. Model results indicate that wastewater-based algal bio-oil can provide an opportunity to increase national biofuel output. The potential production of algal bio-oil can reach to 0.98billiongallon/yr, nearly 20% of advanced biofuel projection as outlined in the U.S. Energy Independence and Security Act (EISA) of 2007. LCA results shows significant variations among different locations, WWTPs, and operational seasons. Although not competitive to conventional fossil fuel in energy efficiency, wastewater-based algal biofuel could offer significant benefit in controlling GHG emissions. However, spatial analysis shows that only 61% of the total wastewater could be used, based on current land use efficiency for algae cultivation and land availability around each WWTP in a radius where algal biofuel production is energy positive (energy output>energy input). These results indicate that land availability could be a significant challenge for wastewater-based algal biofuels that have not been considered in previous studies. They also suggest that improvement should be made in technological development and system design to increase energy and land use efficiency for full potential of wastewater as a promising resource for algal biofuel production. Although focusing on the U.S. as the case study, the developed methodology could be used for spatially explicit analysis of algal biofuel integrated with wastewater on macro-scale in other regions as well.