Herein, we develop a simple electrochemical sensor based on the confinement effect of vertically ordered mesoporous silica film (VMSF) and the o-phenanthroline (Phen) chelating agent for ultrasensitive determination of ferrous ions (Fe2+). Fe(Phen)(3)(2+) obtained from the chelation between Fe2+ and Phen, shows higher electrochemical activity and larger geometric dimensions compared with free Fe2+, which could be effectively preconcentrated into the inner nanochannels of VMSF through spatial and electrostatic confinement effects. Differing from the conventional anodic stripping voltammetry, Fe(Phen)(3)(2+) could be enriched by convenient mechanical stirring and further detected by VMSF-modified indium tin oxide (VMSF/ITO), achieving an outstanding detection performance in terms of a linear range (1.0 nM-13.0 mu M) and a limit of detection (LOD, 0.66 nM). Moreover, the established electrochemical sensor can accurately measure total iron and distinguish between different valence states (Fe2+ or Fe3+) with the help of reducing agents. Thanks to the outstanding antifouling ability of VMSF, the sensing platform consisting of VMSF/ITO and Phen chelating agent enables the precise determination of Fe2+ in complex real samples including tap water, pond water, FeSO4 tablets, and even colored samples, which can avoid the tedious sample pretreatment process and be applied in a wide range of scenarios.