At least one-third of the 34 million people living with human immunodeficiency virus (HIV) worldwide are infected with latent tuberculosis (TB). The aim of this study was to determine the rate of HIV infection in TB patients and its determinants in Wuxi City, China. TB patients attending health institutions (12 selected sites) for TB diagnosis and treatment were enrolled in this study. TB diagnosis, treatment and HIV testing were done according to the national guidelines. Blood samples were collected for anonymous HIV testing. Among the TB patients, the HIV prevalence was 13.66% (1493/10,926). Multivariate analysis showed that gender, age, education, marital status, per capita monthly income, patient residence, family size, distance from a health institution, knowledge of HIV–TB co-infection, and knowledge of HIV may be risk factors for HIV–TB co-infection (all: odds ratio > 1, p < 0.05). The prevalence of TB in those with HIV was higher among the study participants. Improving public awareness of HIV–TB co-infection, regularly screening and improving follow-up can reduce the occurrence of HIV–TB co-infection.