The step-feed sequencing batch reactor (SBR), a novel operation mode of SBR, was used for the study of nitrogen removal from real municipal wastewater. The process had achieved advanced and enhanced nitrogen removal by three phases of consecutive aerobic and anoxic periods and also three steps of feeding, combined with real-time control. The control strategies utilized pH and oxidation reduction potential (ORP) as on-line control parameters, especially the derivative of pH, which can flexibly control the durations of aerobic and anoxic phases. Moreover, the relationship of each step influent flow distribution and influent C/N was discussed in this paper, which there was a unique and optimum step ratio ( λ opt) for the wastewater with a given C/N ratio. Based on different influent C/N ratios, there were three different levels of step ratio ( λ), that is, λ < 1, λ = 1, and λ > 1, correspondingly increasing influent flow fill mode, equal influent flow fill mode, and decreasing influent flow fill mode. In this study, when step ratio was 1.5 using real municipal wastewater as influent with C/N ratio of 3.5, the lower aeration consumption and complete denitrification using influent as carbon source could be achieved. Nitrogen removal efficiency could be greatly improved by step feeding in the SBR, total nitrogen in the effluent was lower than 2 mg/L and the average TN removal efficiency was higher than 98%, while only requiring small amount of external carbon source.