This research aimed to characterize Vibrio species from cholera-suspected patients in three hospitals in Dinajpur. We collected 36 stool samples and identified Vibrio spp. using cultural, morphological, and biochemical tests. The highest prevalence was observed in children (18.18%), compared to adolescents (0%), adults (16.67%), and senior adults (16.67%). Cultural identification revealed yellow colonies on TCBS agar, hemolytic colonies on blood agar, pale non-lactose fermenting colonies on MacConkey agar, and white, opaque colonies on nutrient agar. Gram staining showed gram-negative, curved rod-shaped bacteria. Biochemical tests indicated positive reactions for oxidase, catalase, MR, TSI, citrate utilization, MIU, and indole, while VP was negative. Molecular characterization by PCR and gel electrophoresis using 16S rRNA primers confirmed the presence of Vibrio spp. Antibiotic sensitivity testing showed that isolates were sensitive to cotrimoxazole-25µg (21 mm to 30 mm), neomycin-30µg (22 mm), gentamycin-10µg (15 mm), streptomycin-10µg (20 mm), and tetracycline-30µg (17 mm), but resistant to penicillin, mecillinam, and amoxicillin. These findings suggest streptomycin and cotrimoxazole as effective drugs for controlling cholera.