To explore the dynamic changes and genetic characteristics of coxsackievirus B3 (CVB3) isolated from different sources in Shanghai, the full VP1 sequences of CVB3 isolates from environmental sewage surveillance, healthy children and acute flaccid paralysis (AFP) surveillance were amplified and sequenced in this study. Similarity and phylogenetic analysis of CVB3 isolates in Shanghai and global representative CVB3 strains were performed. The results showed that there were 120 CVB3 strains isolated from different sources in Shanghai from 1989 to 2021, belonging to D and E genotypes. The similarities of nucleotide and amino acid of Shanghai CVB3 isolates were from 78.7% to 100% and from 93.3% to 100.0%, respectively. In 2016, the genotype E CVB3 was firstly isolated from sewage in Shanghai, and it was isolated again in 2021. The nucleotides of genotype E CVB3 isolates from sewage in 2021 were similar to those of CVB3 isolates from hand, foot, and mouth disease (HFMD) cases in Guangdong in 2020. But, Shanghai has no reported infection cases of genotype E CVB3. It is still necessary to have a long-term surveillance for sewage and enterovirus cases to improve the sensitivity of enterovirus surveillance, and provide data support for the diagnosis and treatment of enterovirus-related diseases.