Biosensors have been developed to estimate the BOD level of various pollutants. The main objective of this work is to develop a microbial biosensor which can be able to assimilate large number of biological samples and other substrates. The present study reveals that the yeast species Pichia nakasei, Hansenula anomala acted as a new alternate for BOD estimation. To develop microbial biosensor the above pure culture was mixed together in the ratio of 1:1 and immobilized on a special type of matrix and coupled under sandwich model. The constructed biosensor was connected with a BOD meter. The optimum temperature was determined and standardization was carried out using Glucose:Glutamic acid (GGA).Then the assimilation of different substrates (carbohydrate, amino acids, alcohols, organic acids, aldehydes and heavy metal ions) were analyzed. Finally BOD of different types of real samples was determined.
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