A new microbial biosensor was developed by immobilizing Pseudomonas fluorescens cells on eggshell membrane via physical adsorbtion. Bacteria-modified eggshell membrane was fixed tightly onto the surface of a carbon paste electrode (CPE) with a silicone rubber o-ring to construct a microbial biosensor. The measurements were based on the respiratory activity of the cells caused by oxygen consumption during bacterial metabolism. A mediated biosensor was also developed by ferrocene. As well as the response characteristics, stabilities and substrate specificities were investigated. Data were given as the comparison of mediated and unmediated systems.