This study was performed to fabricate a batch-type SPR biosensing system using a thiolated E. coli antibody coupling, and to explore the feasibility of real-time detection of E. coii in a stagnant sample solution. In advance. “O” and “K” antigenic serotype E. coli antibodies were thiolated with sulfo-LC-SPDP and dithiothreitol, and immobilized by chemisorption in the gold surface of compact SPR sensors. When the SPR biosensor immobilized with E. coli antibody monitored a E. coli solution, it took 3 to 5 min to stabilize. The SPR biosensing system developed in this study was able to detect E. coli in the range above 10$^4$ CFU/mL at the 0.05 significant level. Also, the SPR biosensor had possibility to significantly detect E. coli in the range of 10$^2$ to 10$^4$ CFU/mL in E. coli solutions. Meanwhile, when the SPR biosensor immobilized with 5. coli antibody was cleaned with NaOH solutions, its ability to detect E. coli largely decreased due to wash-out of the immobilized antibody. In order to reuse the SPR sensor, it should be antibody-immobilized newly.