The invention discloses an O-substituted dioxazine compound with bactericidal activity, a preparation method thereof and application thereof. In the chemical structural general formula (I), R1 is phenyl or phenyl with substituted group. The preparation method for the compound of the general formula (I) comprises the following two reaction steps: catalytic hydrogenation de-protection and N-acylation reaction, wherein the first step reaction comprises that the benzyl protecting group nitrogenized in A is removed, the second step reaction comprises that different side chains are connected in the diphosgene reaction so as to obtain the compound of the general formula (I), and the reaction formula is characterized in that the R1 in the reaction formula is the same as the R1 in the general formula (I). The bioactivity test shows that the compound of the general formula (I) at 500 mg/L has 70 to 100 percent of bactericidal activity for Erysiphe graminis.