Abstract: Biofilms and surface-associated microbial cells present public health risk and corrosion hazards in different environments including living tissues, indwelling medical devices, water distribution systems, and natural aquatic systems. Enhanced growth of biofilms provides niche conditions capable of harboring pathogens. Despite intense efforts in anti-fouling strategies, effective methods and early warning systems suitable for biofilm detection and bacterial characterization are essentially nonexistent. The objective of this study was to design a portable biosensing device with a flexible fiber optic probe that can be used in the field for in-situ analysis of biofilms. The research was based on cultural and biochemical assays to study different stages of biofilm development on surfaces in various water environments. Microbial assays were performed to compare bacterial cells in different stages of biofilm growths. Biochemical assays were used to quantify bacterial proteolytic, glucosidic, lipophylic, and metabolic activities in biofilms using fluorescence techniques. Substrates used in the quantification of these biochemical activities resulted in fluorescence signals upon their exposure to specific target receptors in biofilms. Pure culture bacterial biofilms were used for signal optimization. Mixed culture biofilms grown on glass coupons were incubated in PVC and cast iron laboratory-scale pilot water distribution systems and used for applicability study. In pure culture biofilms, a linear relationship between the age of biofilm and fluorescence signals of all biochemical activities was observed. Two-month-old mixed culture biofilms grown in both PVC and cast iron laboratory-scale pilot water distribution systems were also evaluated for bacterial biochemical activities. Compared to cast iron, biofilms grown in PVC systems showed an increase of 36, 11, and 6 folds in proteolytic, glucosidic, and lipophylic activities, respectively. Fluorescence signals generated in biofilm samples using specific substrate molecules can be utilized as a platform to design a miniaturized fluorescence biosensor device for non invasive in-situ biofilm detection and characterization.