Vegetable oil-based biofuels are promising carbon-neutral fuels to replace fossil fuels. The study focuses on a comparative analysis of injection and spray characteristics of DF and RO and the relationship between spray and emission formation. Steady nozzle flows were simulated in Ansys Fluent. Free sprays were modeled with 0D spray models, and the relationships between atomization parameters and TKE at the orifice exit were analyzed. Sprays of DF/RO blends in real combustion chamber conditions were simulated with a multi-zone spray model in DIESEL-RK. The influence of RO content on fuel atomization and fuel-air mixture formation were analyzed. Compared with DF, RO has lower injection velocity and TKE at the orifice exit. The empirical correlations of mean droplet diameters and spray angle with TKE were developed by using a complex TKE/ν 0.5 with correlation coefficients above 0.988. The increase of the RO content from 0 to 100% leads to a reduction in spray angle by 24% and an increase in spray tip penetration by 15.5%, SMD by 62.1%, and the mass fraction of fuel injected onto the combustion chamber walls from 17.1 to 42%. A correlation between NOx emissions and SMD was fitted with a correlation coefficient of 0.9968.