A simple and rapid method was proposed for humic substances (HS) determination at microgram levels in natural waters. This assay method is based on the binding of a dye, Toluidine Blue (TB), to HS molecules to produce a dye-HS complex, which causes a decrease in absorbance at 630 nm. This method was calibrated with HS samples with up to a concentration of 40 mg L(-1), which covered the range of dissolved HS concentrations present in natural waters. The detection limit was 0.8 mg L(-1) of HS, and the relative standard deviation of 10 replicate measurements for a 20-mg L(-1) standard sample was 3.5%. From the Langmuir adsorption isotherm theory, the binding equilibrium constant and total number of binding sites at neutral pH were calculated to be (8.17+/-0.42)x10(5) L mol(-1) and N of 1.45+/-0.04 mmol g(-1) HS, respectively. The determination results with five water samples from lake, river and pond were consistent with those measured with the reference methods, demonstrating that this quantification method for HS determination was rapid, sensitive and feasible.