A new microplate analytical procedure is described for the determination of nickel (Ni2+) ions in natural water samples. A lophine analogue fluorescent sensor was synthesized and a spectral study showed a selective fluorescence quenching effect of chemical sensor by Ni2+ under optimized conditions. Density functional theory (DFT) calculations confirmed the formation of a Ni(II)L3 complex obtained by the Job plot. The calculations showed that the fluorescence emission peak of L collapses due to the distortion of L in the complex. The simple and fast microplate procedure allowed us to quantify Ni2+ with a linear response from 1.6 to 40 µg L-1 and a quantification limit of 5.4 µg L-1 without the need of a preconcentration step. The optimized procedure using high-throughput microplate assay has been applied for the determination of Ni2+ in natural water samples with good analytical performances.