A biosensor is an integrated bioanalytical device that employs biological elements, as analyte sensor, coupled to a transducer for signal detection. Biosensor provides sensitive and selective determination of various compounds of analytical relevance. Recently, use of self-assembled monolayer (SAM) is gaining attention as biosensor matrix. SAM provides a simple route to functionalize electrode surfaces for biosensor fabrication. The alarming rise in the rates of heart diseases due to abnormal levels of cholesterol in blood has stimulated public concern about the estimation of cholesterol level in desired sample. The present book highlights the preparation, characterization and application of some SAMs for immobilization of cholesterol oxidase, cholesterol esterase & horseradish peroxidase for development of cholesterol biosensor. The fabricated bioelectrodes were tested for various parameters like pH, temperature, interferents etc. and have been utilized for free and total cholesterol estimation in solution & serum samples. The results indicate that the SAMs are likely to provide useful matrices for the fabrication of commercial cholesterol sensing bioelectrodes.