The invention relates to an immobilized sediment and a preparation method and application thereof. The preparation method comprises the following steps: step one, taking a sediment, freezing the sediment, drying the frozen sediment, grinding the dried sediment, and sieving the ground sediment to obtain a sediment sample; step two, mixing the sediment sample with a sodium alginate solution uniformly and preparing standby mixed liquid; andstep three, adding the standby mixed liquid into a calcium chloride solution at a uniform speed, carrying out crosslinking to form an immobilized sediment. Theimmobilized sediment has the density of 2.1 to 2.5 g/cm<3>, the surface aperture of the immobilized sediment is 5 nm to 200 nm, and the superficial area of the sediment is 180 to 250cm<2>; and the immobilized sediment has the excellent retention ability and is capable of releasing the contaminants in the sediment continuously. The immobilized sediment can be applied to the total sediment and soiltoxicity testing; the testing process does not limit the growth of algae; and the density of the algae can be determined by a spectrophotometer. The method is simple and is operated rapidly; and measurement is sensitive.