Abstract Compared with ordinary diesel, biodiesel has greater viscosity, density, and flash point, especially under medium and low load, which is easy to cause poor atomization and inadequate combustion. In this paper, an in-line six-cylinder inland river ship diesel engine was used as a model to conduct simulation tests under 50% load of propulsion characteristics to explore the effects of different fuel injection pressures on oil-gas mixing, combustion, and emission of fuel blended with a small percentage of biodiesel. The results show that the increase of fuel injection pressure can enhance the average turbulence intensity in the cylinder as well as increase the relative flow velocity between oil and gas, which is beneficial to the mixture combustion. High fuel injection pressure will promote flame propagation, increasing the maximum burst pressure and the average temperature in the cylinder. Compared to the fuel injection pressure of 24 MPa, NOx emission increases by 5.5% and 15.6% respectively at 27 MPa and 30 MPa, and decreases by 10% and 25% respectively by SOOT.