Main observation and conclusion A new chemiluminescence (CL) reagent named 4‐amino‐5‐thiocyanato‐phthalyl‐hydrazine (iso‐luminol‐SCN) is synthesized by the bromide‐mediated substitution reaction of iso‐luminol with sodium thiocyanate in dimethyl formamide (DMF) at room temperature. Strong CL is observed for iso‐luminol‐SCN oxidized by hydrogen peroxide (H2O2) in the presence of Fe2+, with the CL intensity strongly depending on the concentration of Fe2+ and being hardly interferred by other common metal ions. Based on this fact, a flow injection CL method is established for the high sensitive and selective determination of Fe2+. The CL intensity was linearly correlated with the concentration of Fe2+ in the range of 1.0 × 10–8 mol·L–1 to 1.0 × 10–5 mol·L–1 following the equation CLnet = 166.3c + 57.7 (c, ×10–6 mol·L–1; n = 7; R2 = 0.9988). The detection limit is calculated to be 3.2 × 10–9 mol·L–1 Fe2+ based on the 3S0/K principle. The relative standard deviation (RSD) was 3.7% for 1.0 × 10–6 mol·L–1 Fe2+ (n = 11), a support for the precision of Fe2+ measurement. The recoveries of Fe2+ were obtained in the range of 98.8%—101.6% in three real water samples, showing the applicability and reliability by the proposed method. The possible CL mechanism is proposed based on the kinetic characteristic of the CL reaction and the CL spectrum.