Tembusu virus (TMUV) is an important pathogen that causes acute egg drop syndrome in poultry. To investigate the epidemiological status of Tembusu virus in Zhejiang province, we collected clinical samples and sera from the local area from 2010 to 2016. A total of 41 out of the 88 collected tissue samples were identified as TMUV-infected by RT-PCR and were confirmed by sequencing. Six TMUV strains were isolated from TMUV-positive samples, and their complete genome sequences were determined. In addition, 19 E gene sequences amplified from RT-PCR-positive samples were determined. Sequence identity values among the 19 E genes and reference E sequences ranged from 96.8% to 100.0%, and they ranged from 97.3% to 99.9% when comparing the six genome sequences and references. Nineteen sites with amino acid mutations were identified in the E protein of nineteen strains, and these were at positions that are usually conserved in other TMUV strains. Antibodies to TMUV in serum samples were detected by indirect ELISA using recombinant EDIII (domain III of the E protein) as the antigen. The results showed that TMUV-specific antibodies were widely present in duck populations, with positive rates of 17.38%, 21.99%, 26.68%, and 17.79% in 2013, 2014, 2015 and 2016, respectively. The data from this study provide a good understanding of the epidemiology of TMUV infections in Zhejiang, China.