Ammonium is one of the most important inorganic nutrients in many ecosystems. The utility of a sensitive method for the measurement of ammonium (NH4+) in water, soil and plant-derived samples using fluorescence of an o-phthaldialdehyde (OPA)-based compound was evaluated in this study. This method involves less toxic chemicals than a conventional method, requires minimal equipment, and allows for fairly rapid sample processing. This method is not only sensitive for low (e.g., sub-micromolar) NH4+ concentrations, but also applicable for samples with much higher concentrations (> 100 μM NH4+) by using a modified spectrophotometric protocol developed herein. This evaluation demonstrates that this method provides a valuable tool for the measurement of NH4+ in a variety of environmental samples including soil extracts, litter leachates and highly polluted water.