The factors affecting case detection of tuberculosis (TB)/human immunodeficiency virus (HIV) co-infection cases were explored in order to provide evidence-based formulation of a TB/HIV co-infection control strategy in China. Four counties with different modes of HIV transmission and varying cooperation mechanisms between TB and HIV institutions were selected. HIV test among TB patients and TB examination among people living with HIV/acquired immunodeficiency syndrome (AIDS) were conducted. Patients also completed questionnaires on potential factors affecting TB/HIV co-infection. The results showed that 19 (1.7%) were HIV-positive among 1089 cases of people living with active TB who received HIV testing. Conversely, there were 126 (10%) cases of active TB among 1255 cases of people living with HIV/AIDS. Among the newly discovered 145 cases of TB/HIV co-infection, AIDS institutions found 86.9% of these co-infections and 14.1% came from TB institutes. In different registration categories, there were 31 (21.4%) cases of smear positive TB, 104 (71.7%) cases of smear negative TB, and extrapulmonary cases accounted for 10 (6.9%). Gender, occupation, marital status, ethnic group, education, HIV transmission route, CD4 level, and type of TB susceptible symptoms had significant effects on whether HIV/AIDS patients were co-infected with TB disease (P<0.01). Sex, age, ethnic group, occupation, and type of tuberculosis had no significant influence on whether TB patients had HIV infection. AIDS institutions could find more TB/HIV co-infection patients in comparison with TB institutions, particularly in patients with smear negative pulmonary tuberculosis and extrapulmonary tuberculosis. Therefore, a cooperation mechanism should be built up between TB and HIV control systems, and routine TB checks should be conducted for HIV/AIDS patients. In areas where sex or drug use is the major transmission mode of HIV infection, antiretroviral therapy should be expanded, treatment adherence should be improved and patients’ immunity level should be increased in order to decrease TB incidence. Cough and expectoration of more than two weeks, bloody sputum, and recurrent fevers could be revealing symptoms in TB screening of HIV/AIDS patients.