The invention discloses a sampling and sample preparation device for river polluted bottom sediment for biotoxicity test and a working method, and belongs to the technical field of river regulation. The device comprises a sample preparation box body, a position adjusting element, a bottom sediment collecting element, a bottom sediment sample preparation element and a power supply. By arranging thebottom sediment collecting element at the bottom end of the position adjusting element, the bottom sediment collecting position can be automatically adjusted through the position adjusting element, time and labor are saved, the mechanization degree is high, bottom sediment at multiple collecting points can be collected at the same time, the number of collected samples is increased, the collectingefficiency is improved, and the later bottom sediment toxicity detection accuracy is improved; muddy water in a collected bottom sediment sample is subjected to in-situ separation through the bottomsediment sample preparation element, the detection period is shortened, the separated sample is divided into a plurality of samples to be placed in different liquid containing holes, when the sample needs to be moved, only the liquid containing drawer needs to be drawn out. The structure is simple, and the operation is convenient.