Biobutanol has many characteristics that make it a better biofuel than bioethanol, now used in the formulation of gasohol. Despite the remarkable advantages of butanol as a fuel, biobutanol production has several limitations, including low values for final product concentration and the phenomenon of degeneration. In this study, we examined the effect of liquid-liquid extraction for the in-situ removal of butanol from fed-batch reactor fermentation broth using Clostridium acetobutylicum ATCC 824. A non-toxic immiscible solvent, oleyl alcohol, extracted the majority of the inhibitory butanol from the aqueous broth, resulting in a high butanol production. In the batch culture, without solvent extraction, butanol production ceased after 36 h at a concentration of 8.5 g/L. Applying oleyl alcohol as the extraction solvent, about 72% of the total butanol produced was extracted and an total butanol concentration of 18.9 g/L was achieved, which was 222 % higher than that with the controlled traditional batch process. During butanol fermentation, in addition, the populations of C. acetobutylicum and the portion of degenerated cells were monitored by using multiplex real-time qPCR with newly designed primers and probe sets, C. aceto set and DGS set. By combining the extraction and the monitoring techniques, it could be possible to produce butanol stably in high concentration without failures.