Abstract: Freshwater sediment from Eastlake, Wuhan, China, was spiked with humus and copper to obtain a series of different levels of total organic carbon (TOC, 1.47%—5.72%)and total copper (120—2700mg/kg dry weight). In laboratory, humus was added to natural freshwater sediment and stored at 20℃ for 30 days in a volume portion of 1:4 (sediment to water), then samples was spiked with copper and stored for 10 days. These humus and copper-spiked sediments were centrifuged under 3000r/min, 5200r/min, 9000r/min, 10500r/min, 12000r/min, 4℃ and 20min to collected its interstitial water (IW). chemica1 analysis showed that, Cu, Fe, TOC concentrations in IW decreased as centrifugal speed increased, meanwhile Ca somewhat increased but Mn concentration had no apparent correlation with centrifugal speed. According to the above results, the centrifugation method for interstitial water was preferable at: 12000r/min(9000g RCF), 4℃, 20min in study of humus effect on the toxicity of copper in sediment.