Accelerating biofuel production has been promoted as an opportunity to enhance energy security, offset greenhouse gas emissions and support rural economies. However, large uncertainties remain in the impacts of biofuels, particularly, on air quality and human health. Sugarcane ethanol is one of the most widely used biofuels, and Brazil is its largest producer. Here a systematic framework, including emission modeling, air quality simulation, and health impact assessment was developed to quantify direct and indirect air-pollution and health impacts throughout the life-cycle of Brazilian biofuel. It was found Brazilian biofuel with dominant effects of pre-harvesting burning and indirect land-use change has increasing life-cycle emissions (non-GHG) due to expanding sugarcane areas. Current satellite-based approaches may not account for sugarcane burning emissions due to the small scale of these fires relative to satellite resolution and assumptions regarding fuel loadings. Results also suggest that Brazilian ethanol would cause greater health impacts than US conventional gasoline and cellulosic ethanol if current practices of sugarcane harvesting and land-use change management remain. Effective controls of deforestation, better management of pre-harvesting burning practices, and utilization of agricultural residues, e.g. sugarcane trash and bagasse, would be possible solutions to improve air quality. However, the cost and the maturity of technologies might be the issues. The research framework may assist the future determination on the sustainability of other advanced fuels.