As the advantages of larger sampling throughout, simpler sampling preparation and cheaper instrument and reagent cost, immune-sensor is considered as a preferred screening tool for the determination of small molecular organic pollutants. In this study, an evanescent wave multi-channel biosensor for the detection of micro-pollutants in water environment was developed to detect typical micro-pollutants such as 2,4-dichlorophenoxyacetic acid (2,4-D), microcystin-LR (MC-LR), EDCs and antibiotics. Bio-sensors have shown good detection performances with low detecting limit, high frequency, sensitive and can meet the requirements for the detection of drinking water quality. The performances of the evanescent wave multi-channel biosensor was validated with respect to that of conventional high-performance liquid chromatography, and the correlation between methods agreed well (R² = 0.9762). Based on this bio-sensor an automated online optical bio-sensing system was developed. This system has successfully been applied to long-term, continuous determination and early warning for micro-pollutants. Thus, the bio-sensor system paves the way for a vital routine online analysis that satisfies the high demand for ensuring the safety of drinking water sources and water environment eco-system.