2-(5-Bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP) is a highly sensitive chromogenic reagent that can react with most of the transition and alkaline earth metals. The Ni(II)-5-Br-PADAP complex is more stable than other metal–5-Br-PADAP complexes. In the presence of seignette salt, ethylenediaminetetraacetic acid (EDTA) can decompose most of the 5-Br-PADAP complexes with metals except for iron, cobalt, and nickel. Cetrimonium bromide (CTMAB) as a sensitizer for the color reaction forms a ternary complex with nickel and 5-Br-PADAP with maximum absorption wavelength at 561 nm. CTMAB can significantly improve the sensitivity and selectivity of nickel determination, as well as the stability and solubility of compounds. In this study, the determination of trace nickel in natural water samples was performed by flow injection analysis. The calibration lines were established in the range of 0–200 μg/l of nickel (n ≥ 3), and the limit of detection was 0.093 μg/l. The relative standard deviation was 2.55% for the determination of 25 μg/l nickel (n ≥ 20). The recoveries of this method ranged from 91.0 to 101% for environmental water samples. A large amount of aluminum, calcium, cadmium, copper, bicarbonate, magnesium, zinc, and iron, except for cobalt, did not interfere with the determination of nickel.