The effects of injection pressure and ambient pressure on biodiesel spray characteristics were studied by coupling the in-nozzle flow, and a constantvolume combustion bomb experimental platform was built to verify the accuracy of the coupled numerical model. Spray tip penetration (STP), spray velocity, and droplet size/number distribution of biodiesel were calculated at different injection pressures (200, and environmental pressures (0.1, −0.5, −1.0, and −2.0). The sensitivity to injection pressures and environmental pressures was analyzed with particle concentration as the characteristic parameter. With the increase of injection pressure, the STP of biodiesel increased by 5%, which resulted in a faster decrease in the increasing rate of STP (V ). Under the same conditions, with the increase of injection pressure, the axial and radial velocities of fuel droplets gradually rose, and the high-speed area was enlarged, while the number of droplets and Sauter mean diameter (SMD) space generally decreased. Compared with the injection pressure, the environmental pressure significantly inhibited the STP and velocity of biodiesel, and the increasing rate of STP (V ) decreased gradually and the number of droplet particles and SMD rose with the increase of the environmental pressure.