In the current study, bioethanol has been purified and separated from the culture broth using in situ modified bacterial cellulose (BC) membrane with AMPS. To our knowledge, this is the first report for development of BC composite membrane for bioethanol separation from production media. The characterization of the prepared membrane was investigated for morphology and functional groups via scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and Raman spectroscopy, in addition to the determination of their water and ethanol uptake. The obtained data proved the formation of cellulose multilayers in addition to the existence of its specific function groups. The Amicon cell pervaporation system containing the prepared BC/AMPS membrane has been used for the separation of the bioethanol from the culture broth using nitrogen gas pressure, and the results revealed that the BC/AMPS composite membrane is more efficient than the neat BC membrane in the separation process of bioethanol. At 50-psi nitrogen pressure, the best separation factor and flux were recorded as 15.43 and 98.94 g/m².h, respectively, which were accompanied by the elevation of the bioethanol concentration from 1.98 to 3.22 mg/ml before and after separation, respectively. These findings revealed the promising application of BC/AMPS membrane in the field of bioenergy especially the bioethanol separation.