Effects of fuel and ambient gas temperature on the spray and combustion characteristics of soybean biodiesel were studied in a constant-volume combustion chamber. Four different fuels or fuel blends including B0, B20, B50 and B100 were investigated experimentally. The soot mass data were obtained via a new technique called forward illumination light extinction (FILE). The ambient gas temperature was varied from 700 K to 1200 K. To simulate the engine operating conditions, the ambient oxygen concentration and its density were kept at 21 % and 15 kg/m3, respectively. A higher peak pressure is found as the biodiesel content decreases. B20, B50 and B100 have a shorter ignition delay than B0 and the ignition delay decreases with increasing biodiesel content. The liquid penetration decreases with decreasing biodiesel content. Moreover, the integrated natural flame luminosity (INFL) increases with decreasing biodiesel content. Shorter flame (i.e., soot luminosity) duration and a longer delay between start of combustion (SOC) and the appearance of flame are found as the biodiesel content increases. The flame duration also increases with increasing ambient gas temperature for all fuels. Soot is lower and appears later at a lower ambient gas temperature, while it is burned out at around the same time. Near-zero soot mass was observed for all tested fuels at 700 K. A shorter soot formation process is observed for biodiesel fuels. The soot reduction using B20 and B50 is not obvious compared to B0 at a low temperature. But under the ordinary diesel engine operating condition at 1000 K, the soot reduction is significant. It is also found that the soot can be reduced by 60% and above when B100 is used in this study.