Reusable and portable evanescent wave biosensor for rapid, sensitive and highly selective direct detection of adenoviruses was developed. Here, specific antibody for the hexon protein of the virus was used as a recognition element. Linear detection range was determined from 100PFU/ml to 2000PFU/ml. This direct detection system can be regenerated successfully with over 5 times without significant deterioration of the sensor performance. The developed biosensor system has high specificity towards adenovirus, and it was applied to environmental waste water samples for the detection of adenovirus with 95 to 117% recovery and 1.1 to 8.1% cv values (n=2).