The composition of biodiesel is significantly influenced by the feedstock sources. This variation in biodiesel composition may cause changes in fuel physiochemical properties and further affect the engine operation processes such as the fuel injection and spray processes. In this study, the macroscopic and microscopic spray parameters of diesel fuel and three biodiesel components (methyl laurate, methyl oleate and ethyl oleate) on a diesel common rail injection system are investigated at conditions of different injection pressures and ambient pressures. The macroscopic spray parameters, including the tip penetration distance, projected spray area, spray front velocity, spray cone angle, and the maximum spray width, of different fatty acid esters are characterized and compared to those of diesel fuel. Further, the images of fuel spray development obtained by the high speed camera are converted to grayscale ones, and the grayscale values within the spray contours are extracted to evaluate the light intensity level and distribution characteristics for different fuels. Finally, the microscopic spray characteristics of test fuels measured using a split laser particle size analyzer indicated that fuel properties also play an important role in the droplet sizes, and the droplets of methyl laurate and diesel have smaller SMD than methyl oleate and ethyl oleate.