3,4-Dichloroaniline (3,4-DCA) is a main intermediate of many fine chemical products, it is easy to remain in production wastewater, which is highly toxic and difficult to biodegrade. In this experiment, Gram-negative bacteria were isolated from dye wastewater, which could grow with 3,4-DCA as the sole carbon source and energy source. Two strains with great morphological differences, P11-1 and L13, were screened to construct mixed bacteria, which were used in the transformation test of 3,4-DCA. By optimizing the proportion of the mixed bacteria, the transformation rate reached 73.33%, which was significantly higher than that of single bacteria. The two strains were identified as Pseudomonas aeruginosa by observing the morphology of the strains and determining the 16S rDNA sequence. Through the single factor test to investigate the transformation ability of the mixed bacteria to 100 mg/L DCA in 3 days, and further optimizing the transformation conditions by orthogonal experiments, it is concluded that the best combination of the conditions for the transformation of the mixed bacteria is as follows: inoculums concentration 3% (volume fraction), 30 ℃, pH 8.0, shaking velocity 190 r/min. Under these conditions, the transformation rate is up to 89.26%, in which the pH value has the greatest effect on the transformation ability of the mixed bacteria. GC-MS analysis showed that the main metabolite of the mixed bacteria was 3,4-dichloroacetanilide. Enzyme activity analysis showed that the mixed bacteria had higher N-acetyltransferase (NAT) activity, that is, it detoxified 3,4-DCA by acetylation.