Abstract: Biofuels have extensive available resources and have an immense potential as promising alternative fuels for automobile. The application advantages of biofuels are mainly reflected as particulate matter (PM) reduction, carbon neutral, greenhouse gases reduction, waste utilization, energy and economic security, and fuel pluralism. Based on the understanding of molecular structure effects of biofuels on soot formation and particles morphology, the effects of alcohols, ethers, esters and biodiesel on spray and combustion process in constant volume bomb in recent years are retrospectively analyzed in this paper. For the mixture, macromolecular ester fuels and polyoxymethylene dimethyl ether (PODE) are conducive to the improvement of liquid spray, while biodiesel, small molecules, dimethyl ether (DME) and alcohols are reversed. Alcohols are advantageous to the extension of mixing time and the increasing of vapor-phase mixture. Through the influence integrated assessment, alcohols show the best performance on the spray, atomization and combustion, while biodiesels show the worst. But in terms of combustion, PODE is the best choice without considering spray and atomization. For binary alternative-diesel fuel blends, methanol or butanol is the best additive based on synthetically considerations on spray, atomization and combustion. To meet the requirements of the fuel application of diesel engine, ternary fuel or even quaternary fuel have been proposed and explored. This review can help to form a systematic understanding on fuel recombining and obtain the guide of clean and efficient fuel formulation for diesel engine.