Strobilurin fungicides are widely used in rice and corn planting due to their broad-spectrum and fungal mitochondrial inhibitory activities. However, their environmental residues will cause damage to eukaryotes such as chlorella, Daphnia magna and zebrafish, etc. In this study, a bacterial strain B1 was isolated and identified as Hyphomicrobium sp., which could degrade trifloxystrobin, a representative of strobilurin fungicides. A novel esterase gene triH responsible for the trifloxystrobin detoxification and trifloxystrobin acid generation was cloned. TriH shared low identity with the reported alpha/beta-hydrolase superfamily esterases (only 26–28%). The Km and kcat values of TriH for trifloxystrobin were 2.35 ± 0.24 μM and 48.66 ± 6.43 s−1. Also, TriH could catalyze the de-esterification reaction of azoxystrobin, picoxystrobin, and kresoxim-methyl to generate the corresponding daughter acid and methanol. Strain B1 can effectively remediate the trifloxystrobin-polluted freshwater and protect chlorella, Daphnia magna and zebrafish from the harm of trifloxystrobin. This work offers a potential strain as well as an enzyme for the remediation of strobilurin fungicides-contaminated environments.