The invention relates to a method for determining chloramphenicol in freshwater pond sediment by applying a liquid chromatograph/mass spectrometer. The method comprises the following steps of firstly, collecting a sediment sample of an aquaculture pond; secondly, extracting the sample; thirdly, purifying with n-hexane; fourthly, extracting water saturated ethyl acetate solution; fifthly, blowing extracting solution to be dry with nitrogen gas, and dissolving the dry extracting solution with acetonitrile-water (5+95) to obtain standby solution; sixthly, performing solid phase extraction column for eluting; seventhly, extracting the water saturated ethyl acetate solution, and blowing dry with the nitrogen gas; eightly, dissolving methanol water (50+50), whirling, filtering by a microfiltration membrane of 0.22[mu]m and determining by using the liquid chromatograph/mass spectrometer. According to the method disclosed by the invention, by pretreatment for the freshwater pond sediment, a matrix disturbing substance is removed, the sensitivity is improved, and the pretreatment process is optimized; in addition, the sample is prevented from being derivaed, so that the operation step is simplified; a reliable detection means can be provided for checking a pollution source and evaluating the influence of the ecological environment on healthy breeding.