To improve the performance and emission characteristics in the diesel/CNG dual-fuel combustion mode, bio-fuel of n-butanol as the additive in the pilot fuel was investigated by sweeping a wide range of CNG substitution rates. The experiments were conducted at fixed CA50s of 4.5 and 5.5 °CA ATDC for approximately 5 bar IMEP (low load) and 7.5 bar IMEP (medium load). At low load, the pilot fuel with n-butanol can shorten the combustion duration for each CNG substitution rate, and D90B10 (90% diesel/10% n-butanol by volume basis as the pilot fuel) can improve the indicated thermal efficiency (ITE) with the relatively high CNG substitution rates and reduce CO emission. D80B20 (80% diesel/20% n-butanol by volume basis as the pilot fuel) can reduce both the NOx and CO emissions simultaneously compared to pure diesel as the pilot fuel. At medium load, the pilot fuel with n-butanol can improve ITE with the relatively low CNG substitution rates. It can also reduce the CO emission but slightly increase the NOx emissions. It can be found that the THC emissions are more sensitive to the CNG substitution rate. In addition, the pilot fuel with n-butanol can reduce the CO2 emission at most CNG substitution rates.