Objective: To investigate the status and influencing factors of latent tuberculosis infection (LTBI) among enterprise employees in Wenzhou City, Zhejiang Province, so as to provide evidence for optimizing tuberculosis prevention and control strategies in enterprises. Methods: From August 2024 to September 2025, a convenience sampling method was used to select employees from 26 enterprises in Wenzhou City as the study participants. Demographic information, enterprise scale, and industry type were collected by reviewing enterprise employee management files. Interferon-gamma release assays were used to detect Mycobacterium tuberculosis infection and screen for LTBI. Factors affecting LTBI among enterprise employees were identified using a multivariable logistic regression model. Results: A total of 6 476 participants were surveyed, including 3 674 males (56.73%) and 2 802 females (43.27%). The median age was 37.00 (interquartile range, 20.00) years. A total of 994 cases of LTBI were detected, with a detection rate of 15.35%. Multivariable logistic regression analysis showed that male (OR=1.397, 95%CI: 1.211-1.612), age ≥45 years (45-<60 years, OR=1.919, 95%CI: 1.649-2.234; ≥60 years, OR=4.425, 95%CI: 3.170-6.177), household registration outside Zhejiang Province (OR=1.928, 95%CI: 1.577-2.357), employment in small or medium-sized enterprises (medium-sized, OR=1.663, 95%CI: 1.392-1.988; small-sized, OR=1.665, 95%CI: 1.358-2.040), and working in the manufacturing industry (OR=1.799, 95%CI: 1.147-2.822) were associated with a higher risk of LTBI among enterprise employees. Conclusions The detection rate of LTBI among enterprise employees in Wenzhou City was relatively low. Male employees, those aged ≥45 years, those with household registration outside Zhejiang Province, those working in small or medium-sized enterprises, and those in the manufacturing industry were identified as high-risk populations for LTBI.