In this study, a new microbial biosensor based on Bacillus subtilis was developed for sensitive determination of paracetamol. The microbial biosensor was modified by using carboxylated multiwalled carbon nanotube (cMWCNT) and conductive polymer, polyaniline (PANI) in presence of glutaraldehyde on a gold working electrode. Paracetamol measurements by the microbial biosensor were carried out at 0.5 V applied potential with amperometric method. In the optimization studies of the microbial biosensor, the effect of (amount or concentration of) the bioactive layer components such as MWCNT, PANI, Bacillus sp. were carried out. In addition, working conditions of the microbial biosensor such as pH and temperature were also investigated.