The film doped with tris-(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) ([Ru(dpp)3]2+) as an oxygen quenching indicator exhibited a good linear relationship, fast response time, long-term stability, and enhanced sensitivity to dissolved oxygen after optimizing the sol-gel processing parameters. Fiber-optical microbial sensors for the determination of biochemical oxygen demand (BOD) were described. The sensing films consist of two layers of an oxygen-sensitive fluorescent material, and three different kinds of seawater microorganisms immobilized in poly(vinyl alcohol) sol-gel matrix. The fluorescent properties and the response behaviors of the film were investigated. The effects of temperature, pH and sodium chloride concentration on the sensing films were studied as well. For low biochemical oxygen demand, the film of sieved bacteria from seawater was superior in respect of sensitivity and is expected for further development.