Background:　The　demethylation　potential　of　environmental　pollutants　is　possibly　an　innate　part　of　their　comprehensive　health　risk.　This　paper　develops　a　novel　method　called　TDQ　to　quantify　the　demethylation　epigenetic　toxicity,　termed　the　5-AZA-CdR　demethylation　toxic　equivalency,　of　aquatic　samples　from　the　heavily　polluted　Bohai　Bay　using　Hep　G2　cell　lines　transiently　transfected　with　the　pEGFP-C3　plasmid　containing　a　methylated　promoter　of　the　EGFP　reporter　gene　inserted　artificially　in　vitro.　Results:　If　the　aquatic　sample　extract　has　strong　total　demethylation　potential　to　the　promoter,　its　methylation　level　will　decrease,　and　increased　green　fluorescence　will　be　observed　under　microscopy　after　TDQ　co-incubation.　The　5-AZA-CdR　was　selected　as　a　representative　demethylation　agent　to　validate　the　principle　of　the　TDQ　method　on　three　levels:　significant　dose-response　relationships　between　the　concentration　of　5-AZA-CdR　and　the　methylation　level　of　promoters,　mRNA　expression　level　of　the　EGFP　gene,　and　the　fluorescence　intensity　of　EGFP　proteins.　Twenty　extracts　from　aquatic　samples　are　successfully　quantified　with　the　TDQ　test.　Eight　of　them　return　meaningful　results　ranging　from　0.00004　to　0.20053　mu　M　5-AZA-CdR　toxicity　equivalents.　Conclusions:　The　TDQ　method　is　a　reliable　and　rapid　assay　for　the　quantification　of　the　DNA　demethylation　potential　of　aquatic　sample　extracts,　which　may　shed　light　on　the　safety　evaluation　of　food　material.