Abstract: Four strains of high-efficiency nitrobacteria, F1, F4, Y2 and Z1, were isolated from one fishpond’s silt. The Sherlock microbial identification system (MIS) was used to perform the whole cellular fatty acid composition analysis and identification for the four strains, combined with physiological and biochemistry indexes analysis. They were preliminarily identified as Rhodococcus (F1 and F4), Pseudomonas (Y2) and Micrococcus (Z1). The test of nitrifying rate showed that the nitrifying rate of F1, F4, Y2 and Z1 reached 3.51, 3.43, 3.46 and 3.52 mg/(L·d), respectively. The four strains were mixed equally to form a bacterial complex and the orthogonal experiment under comprehensive cultivation conditions by changing cultivation conditions was carried out. The results showed that the highest NH₄⁺-N conversion rate of 70.02% was obtained at 35℃, pH 8.5, with 20 mL medium content and 25% inoculum size. Factors affecting nitrification effectiveness were in the order of medium content > temperature > pH > inoculum size.