The discovery of complete ammonia oxidizer (comammox) was a breakthrough in the study of nitrification. However, slow growth of comammox bacteria makes it challenging to distinguish them from traditional ammonia oxidizing microorganisms. Genomic data indicated that comammox bacteria encoded genes that can metabolize urea and had higher nitrite tolerance, which could only be found in several ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). This implies that using nitrite and urea as nitrogen sources may accelerate comammox bacteria's enrichment efficiency. In this study, two reactors using nitrite and urea as substrates, respectively, were operated for 390 days. At the end of cultivation, the reactor fed with urea exhibited higher nitrification potential than the reactor fed with nitrite. Comammox bacteria outcompeted AOA and AOB, regardless of whether they were cultured with nitrite or urea. Using nitrite can improve the proportion of comammox amoA to total amoA of 92%, while using urea may increase the proportion of comammox bacteria among total bacteria to 14.2%. Metagenomic results implied that nitrite was converted to ammonia by nitrate reduction and absorbed by comammox bacteria. On the other hand, urea may be directly utilized as substrate. These results demonstrated that using different nitrogen sources caused niche differentiation of comammox bacteria, AOA, and AOB. Using nitrite can increase the relative abundance of comammox amoA to total amoA, while using urea can increase the quantity of comammox amoA. Comammox bacteria were dominant among ammonia oxidizing microorganisms for both nitrite and urea cultures.