The invention relates to a method and a kit for extracting total desoxyribonucleic acid in crude oil. The method comprises the following steps of carrying out pretreatment on the crude oil; cracking a viable cell and/or a dead cell by a positive-negative surfactant method to obtain a supernatant fluid containing the total desoxyribonucleic acid; extracting the supernatant fluid to obtain the total desoxyribonucleic acid by utilizing one of four purification methods of chloroform-alcohol precipitation, phenol/chloroform-alcohol precipitation, a chloroform-purification column and a phenol/chloroform-purification column. According to the method and the kit, aiming at the self property of the crude oil and the characteristic that components are complicated, four methods and kits for extracting the total desoxyribonucleic acid in the crude oil, which are wide in scope of application, are developed; the total desoxyribonucleic acid in the crude oil can be effectively extracted.