The invention discloses a method for removing the bio-toxicity of wastewater generated in chemical synthesis of vitamin B1. A three-dimensional electrolysis-Fenton catalytic oxidation method is adopted to preprocess wastewater efficiently and physically. (1) Organic matters are decomposed by following ways: pollutants are directly oxidized by hydroxyl free radicals (OH.), which are generated on the surface of a titanium alloy coated electrode; and pollutants are indirectly oxidized by Fenton oxidation between H2O2 generated through reduction reactions of a negative electrode and added FeSO4.7H2O. (2) A reaction tank is made of a shell, a plurality sets of parallel positive electrode plates and negative electrode plates, a filled ionic electrode, a direct current stabilized power supply, aninlet water distributor, a bottom aerator, an upper slag removing tank, and an outlet water distributor. A titanium alloy coating electrode is taken as the positive electrode, a stainless steel plateis taken as the negative electrode, and a modified carbon based filling material with a large contact resistance is taken as the ionic electrode. (3) By adopting the provided method, the COD removalrate can reach 50% or more, the B/C ratio is increased to 0.4 or more, and the bio-toxicity is greatly reduced.