The invention discloses a preservation method of sediment in river and lake on the basis of an oxygen isolation protective layer. In the method, a preservation apparatus is composed of an inner-layer self-sealing bag, a sediment protective layer and an outer-layer self-sealing bag. The preservation apparatus is simple in structure and is convenient to use. By means of the inner-layer self-sealing bag and the sediment protective layer, a sediment sample used for experiment in the inner layer is prevented from contacting with oxygen, which causes some components being easy to oxidize in the sediment protective layer to cause conversion, thereby ensuring the authenticity and the scientificity of data from the analysis of the sediment sample used for experiment. The self-sealing bags are low in cost, are easy to buy and are suitable for being used in a large amount on-site. The method can solve a problem of oxygen-isolation preservation of the sediment in river and lake (especially the sediment requiring long-distance transport).