The aims of this study were to extract a tylosin‐degrading enzyme from Burkholderia vietnamiensis and to investigate the enzymatic degradation of tylosin and metabolic product formed. The results showed that the enzyme was extracted and purified to homogeneity with 23.5‐fold purification and a specific activity equal to 247.1 U mg−1 protein. This enzyme with a concentration of 1000 U mL−1 could degrade 100% of 200 mg L−1 tylosin after 180 min of incubation at 35°C. The toxicity of metabolites was lower than that of parent compound. The degradation kinetics followed an empirical model of the natural logarithm of concentration versus the natural logarithm of time, and the calculated half‐lives were about 3.54–3.91 min when the initial concentrations of tylosin ranged from 200 to 600 mg L−1. The enzymatic reaction of tylosin degradation could be well described using Michaelis‐Menten equation, and values of Michaelis constant and maximum velocity were 326.91 µmol L−1 and 16.08 µmol L−1 min−1, respectively. High‐performance liquid chromatography and mass spectrum indicated that tylosin was degraded into two metabolites. The pathway of enzymatic degradation might be that tylosin A was firstly transformed to tylosin B, and then the hydrolysis of lactone bond and the reduction of aldehyde group occurred. These results may provide the basis for biotreatment and removal of tylosin residue in solid and liquid wastes. © 2017 American Institute of Chemical Engineers Environ Prog, 36: 879–886, 2017