Objective:To establish an optimal method of template preparation for sequencing avian influenza virus (H7N9) genome. Methods:Sequencing templates were prepared by using random hexamers primers to synthesize single strand cDNA and double strands cDNA;by using influenza virus specific-U12 primer to synthesize single strand cDNA and using random hexamers primers to obtain double strands cDNA;or by using influenza virus specific-U12 primer to synthesize single strand cDNA and using influenza virus gene-specific primer to obtain double strands cDNA. Sequencing library was constructed by using above template and sequencing. After analysis of the data,the results were compared with Sanger sequencing. The best method for template preparation was selected for high throughput sequencing. Results:The cluster pass filter,coverage,single nucleotide polymorphisms (SNPs),and median length were the best in using influenza virus specific-U12 primer to synthesize single strand cDNA and using influenza virus gene-specific primer to obtain double strands cDNA compared with other two methods. Conclusion:High accuracy,short turnaround times,relatively low cost and high throughput make this method becoming possible for surveillance of H7N9 viruses evolution.