Display omitted •A novel approach for anammox bacteria culture was developed based on microfluidics.•Both free and granular forms of anammox bacteria could be cultured on the chips.•Anammox bacteria could grow quickly and be enriched on the chips.•The reaction kinetics of granular sludge with different sizes were obtained.•Microfluidic chip can be a powerful tool for anammox bacteria study. Anammox bacteria are being increasingly investigated as part of an emerging nitrogen removal technology. However, due to the difficulty in culturing, current understanding of their behavior is limited. In this study, anaerobic microfluidic chips were used to study anammox bacteria, showing great advantages over reactors. On-chip fluorescence in situ hybridization (FISH) showed the relative abundance of free form anammox bacteria increased by 56.1 % after one week’s culture, an increase that is three times higher than that of bioreactor (17.1 %). For granular form cultures, the nitrogen removal load reached 2.34 ∼ 2.51 kg-N/(m3·d), which was also substantially higher than the bioreactor (∼1.22 kg-N/(m3·d)). Furthermore, studying the kinetics of nitrite inhibition of granular sludge with different particle sizes (100–900 μm) showed that the maximum ammonia load and the nitrite semi-saturation coefficient noticeably decreased for smaller particle sizes. These results illustrate the usefulness of the microfluidic method for in-depth understanding anammox process and its implementation.