To investigate the spray and combustion characteristics of a ternary blend of butanol-biodiesel-diesel under working conditions similar to diesel engines, a constant volume chamber with optical access coupled with high speed imaging were applied. The spray penetration lengths for fuels with different blending ratio at various ambient temperatures were recorded. A repeatable sudden drop of spray penetration length was observed at 800 K and 900 K for the ternary blends, but not at 1000 K and 1200 K, which was attributed by micro-explosion. Further studies focus on micro-explosion phenomena under non-combusting environment. High speed imaging showed that the tip of the spray jet erupted into a plume sometime after injection for the butanol-biodiesel-diesel blend at initial temperature 1100 K. The phenomenon was not seen with the biodiesel-diesel blend, neither at lower ambient temperature of 900 K. With biodiesel content increasing, micro-explosion becomes excited and more violent. It was concluded that micro-explosion can occur under particular conditions for the butanol-biodiesel-diesel blend, and the results was consistent with previous theoretical study in the literature.