Abstract: Objective To establish a high throughput half-automated nucleotide extraction and purification system to be used in pathogen surveillance network. Methods We used Perkin Elmer automated workstation and disposable tips to establish a half-automated nucleotide extraction and purification system. The cross-contamination and efficiency of the system were compared with different parameters. Results Move speed of the plunger and the relationship between tips and liquid surface are key factors that influence cross-contamination. The concentration of the broth culture was the key factor that determines DNA quantity and quality. Compared with manual method, this half-automated system improved the efficiency by 2.5 folds. Conclusion We establish a high throughput half-automated nucleotide extraction and purification system, which could be integrated into the molecular subtyping working pipeline of pathogen surveillance network laboratory.