Background: Human exposure to Benzene has been associated with arrange of acute and long – term adverse health effect and disease. Methodology: A population based matched case control that comprises of 45 apparently healthy benzene workers as a cases and 35 non benzene workers analyzed as controls was conducted at Khartoum state –Sudan. Blood samples were drawn from participants and serum testosterone was measured by ELISA technique. Data was analyzed by SPSS v16 Results: Serum testosterone levels were showed no significant difference in benzene workers compared to non benzene workers. There is no correlation between serum testosterone level and duration of exposure on benzene workers. Conclusion: It can be concluded that exposure to benzene had no effect in serum testosterone levels on petroleum stations workers.