A microbial biosensing system for detection of hydrogen sulfide has been developed by using immobilized Thiobacillus thioparus TK-m in poly vinyl alcohol matrix, together with a dissolved oxygen sensor. Parameters of immobilization (poly vinyl alcohol concentration and amount of wet cell) were optimized by using statistical software. The obtained values for concentration of poly vinyl alcohol and wet cell weight were 11.3% (w/v) and 45 mg, respectively, where the response time of biosensor was 80 s. Calibration of oxygen concentration based on hydrogen sulfide concentration was investigated between I mg/L and 20 mg/L. The effect of pH and temperature were investigated in specific range of experimental conditions as well. Some parameters including operational stability and detection limit were studied in detail for characterization of biosensing system. In order to determine the operational stability, bio-sensing system at optimized working conditions was used to distinguish viability of microorganisms in polymer beads in period of time. (C) 2014 Elsevier Ltd. All rights reserved.