A sensitive method to detect hemoglobin (Hb) by flow injection (FI) coupled with chemiluminescence (CL) detection was described, which based on Hb strong enhancing effect on weak luminol-hydrazine CL system in alkaline medium. Parameters affecting the CL detection conditions and FI-CL system were optimized. The effects of possible coexisting substances in human blood and serum of detection Hb were evaluated. Under the optimum conditions, the net CL intensity versus Hb concentration was linear in the range of 5.0×10-9～6.0×10-5 g·mL-1 with the detection limit of 5.8×10-10 g·mL-1 (9.0×10-12 mol·L-1). The relative standard deviations (RSDs) for 8 replicate determinations of 5.0×10-7 and 3.0×10-6 g·mL-1 Hb were 1.6% and 1.5%, respectively. In addition, the recoveries of Hb in human blood and serum were carried out and varied from 83.0% to 101.0%. The proposed method has been successfully applied for the determination of Hb in healthy human blood and serum. The possible mechanism of Hb enhancing the weak CL emission of luminol-hydrazine system in NaOH solution was discussed by fluorescence spectrophotometer and UV-Vis spectrophotometer.