The residues of aniline and its derivatives are serious environment pollutants. Aniline dioxygenase (AD) derived from aerobic bacteria catalyzes the conversion of aniline to catechol, which has potential use in the bioremediation of aromatic amines and biorefining process. AD contains four components: a glutamine synthetase (GS)-like enzyme, a glutamine amidotransferase (GAT)-like enzyme, oxygenase, and reductase. ADs from diverse hosts exhibit different substrate specificities against aniline derivatives. However, what component of AD determines AD's substrate specificity is still unknown which limits the effects of extending AD's substrate spectrum through mutagenesis. Here, each component of two ADs (AtdA1A2A3A4A5 and AdoQTA1A2B) which have different substrate ranges was heterologously expressed and purified. The activity of both ADs was successfully constructed in vitro using the purified components. To identify the component that affects the substrate specificity of the ADs, the substrate specificity of each component was studied. The inability of AtdA1A2A3A4A5 to catalyze 4-methylaniline was determined with GS-like enzyme AtdA1; its inability to convert 2-isopropylaniline was caused by the oxygenase component, and its inability to convert 4-isopropylaniline was caused by both GS-like enzyme AtdA1 and oxygenase components. The inability of AdoQTA1A2B to catalyze 2-methylaniline was determined by GS-like enzyme AdoQ; its inability to convert 2-isopropylaniline was caused by both GS-like enzyme AdoQ and oxygenase components. Together, these results show that GS-like enzyme and oxygenase but not GAT-like enzyme or reductase play dominant roles in the substrate specificity of AD, and this finding will facilitate the engineering of AD to expand its substrate range.