Objective To detect pollution status of non_01 Vibrio cholerae in Haihe river by Vibrio cholerae outer membrane protein gene (ompw) PCR technology. Methods A pair of Vibrio cholera ompw primers was synthetized to detect non_01 Vibrio cholerae in 102 water samples collected from 5 sampling sites in Haihe river by ompw PCR approach and the results were compared with that of the routine bacterial separation culture method. When amplified by PCR, isolated non_01 Vibrio cholerae strain Vbo35 was used as positive reference, Shigella, E.coli and Salmonella strains were used as negative reference in the study. Results The total positive rate of Vibrio cholerae in 102 water samples was 52% and the amplified products were 588 bp. The detection results of the PCR approach were 100% in accord with that of the routine bacterial separation culture method. Conclusion The results indicated that ompw PCR technology was a highly sensitive and specific method to determination of non_01 Vibrio cholerae in Haihe river water system, it shortened the determination time 4～5 times, and could provided a useful determination method for non_01 Vibrio cholerae.