Disclosed is an in-situ detoxification method for a heavily contaminated site by hexavalent chromium. The method includes injecting a chemical reducing agent into a site heavily contaminated with hexavalent chromium, and injecting a chromium soil remediation microbial agent into the site after 0-10 days. The in-situ detoxification method includes: first, in-situ injecting the chemical reducing agent into the soil, preliminarily reducing high-concentration hexavalent chromium such that the hexavalent chromium of high concentration has no toxic effect on the subsequent ecological microbial agent, then in-situ injecting the ecological microbial agent prepared from biogas residue, a carbon source and sulfate into the chromium-containing soil under pressure, and forming a large-scale reducing buffer zone containing sulfate-reducing bacteria and sulfide in a special maintenance manner in the subsurface soil of the site to reduce the hexavalent chromium continuously and effectively.