Abstract: A method for validation of site-specific water quality criteria of a river basin includes steps of: analyzing the biota distribution characteristics of a river basin; eliminating non-resident related species from the toxicity database of target pollutants; supplementing toxicity values of site-specific resident sensitive species and endemic species; establishing a resident toxicity database for the river basin; comparing the degrees of fitting of species sensitivity distribution (SSD) models and identifying the optimal SSD model; and validating the water quality criterion value to complete the derivation and validation of the site-specific water quality criteria of the river basin. The method can improve the nativeness of the site-specific water quality criteria of the river basin, be used for the formulation of the site-specific water environment quality standards of the river basin, and provide support for the water quality management of the river basin.