The invention discloses a holothurian aquaculture water modifier for removing impurities and inhibiting harmful microbe propagation with a one-step method, and a preparation method and application of the holothurian aquaculture water modifier. Aiming at physiologic nature and cultivation characteristics of holothurian, and through use of the marine microbial technology, the holothurian aquaculture water modifier comprises bacillus flexus CGMCCNo.5115, bacillus subtilis CGMCCNo.5116 and bacillus cereus CGMCCNo.5117, which are separated and screened from the holothurian culture environment. The preparation method comprises the following step: using corn flour and soya bean flour as main culture materials, fermenting the materials at 28-37 DEG C for 24-48 hours, using a medical stone and rice husks as adsorption and suspension carriers, and processing to obtain the holothurian aquaculture water modifier for removing impurities and inhibiting harmful microbe propagation with the one-step method. The holothurian aquaculture water modifier is beneficial to the three-dimensional purifying effect of the marine aquatic water, can adsorb hazardous substances of heavy metal ions such as Hg2+, Pb2+, Cr6+, Cd2+ and Cu2+ in the water, greatly reduces the content of hazardous substances such as ammonia nitrogen, nitrite and hydrogen sulfide, deodorizes by using flowing water, effectively inhibits pathogenic microorganisms, prevents holothurian diseases, and improves yield and quality of holothurian aquaculture.