The control of drinking water quality is critical in preventing fluorosis. This study found that when humic substances (HS), typifying natural organic matter (NOM) in water, aggregates the response of fluoride ion selective electrodes (ISE) may be perturbed. Dynamic light scattering (DLS) results of both synthetic solutions and natural water sample suggest that HS aggregation is affected by HS origins and water chemistry. Low pH and high ionic strength are most favourable for HS aggregation. Fluoride concentration in the presence of HS aggregation has a reduction up to 19%. A new “open cage” concept has been developed to explain this reversible phenomenon. The interference of HS aggregation on fluoride measurement can be effectively removed by centrifugation pre-treatment.