Interaction between humans, livestock, and wildlife plays an important role in zoonotic disease transmission. The emergence of Leptospira in humans, rodents, and cattle remains relatively understudied. A cross-sectional study was conducted between February and May 2023 in Muheza to determine evidence of Leptospira infection and associated risk factors in rodents, cattle, and humans. A total of 479 serum samples from rodents (n=201), humans (n=198), and cattle (n=80) were examined by using the Microscopic Agglutination Test (MAT) to detect antibodies against six live Leptospira stock culture serovars, including Pomona, Hebdomadis, Canicola, Grippotyphosa, Sokoine, and Lora. Additionally, a questionnaire survey was conducted on 140 respondents to establish potential risk factors for transmission of Leptospira. The overall Leptospira seroprevalence in rodents, cattle and humans was 6.0% (12/201; 95% CI: 3.12%-10.20 %), 12.5% (10/80; 95% CI: 6.16%-21.79%) and 13.13% (26/198; 95% CI: 8.76%-18.65%), respectively and the most predominant serovars were Grippotyphosa, Sokoine, and Hebdomadis. A significant difference in the seroprevalence was observed in the occupation category, with farmers likelier to be infected with leptospirosis than those in other categories (χ ²= 9.19, df = 3, p = 0.027). This study showed co-agglutination between rodents, cattle, and humans with serovars Hebdomadis, Sokoine, and Grippotyphosa. People aged 36-59 had the highest seropositivity, suggesting they are the most at-risk group. Our study shed light on pathogenic serovars circulating among humans, rodents, and cattle and their associated risk factors. It also highlights the importance of employing a One Heath approach in controlling zoonotic disease.