Leptospirosis is a neglected worldwide zoonotic bacterial disease with a high prevalence in subtropical and tropical countries. The prevalence of Leptospira spp. in humans, cattle and dogs is unknown in Bhutan. Therefore, we sought to find out whether humans, cattle or dogs had been infected in the past with leptospires by measuring antibodies in the serum. We therefore collected blood from 864 humans ≥13 years of age, 130 bovines and 84 dogs from different rural and urban areas in Bhutan and tested the serum for antibodies specific for leptospires with a screening of enzyme-linked immunosorbent assays (ELISA) and a confirmatory microscopic agglutination test (MAT). In humans, 17.6% were seropositive by ELISA and 1.6% by MAT. The seropositivity was stronger in bovines (36.9%) and dogs (47.6%). “Having had a fever recently” (OR 5.2, p = 0.004), “working for the military” (OR 26.6, p = 0.028) and “being unemployed” (OR 12.9, p = 0.041) (reference category = housemaker) were statistically significantly associated with seropositivity when controlled for the effects of other risk factors. However, due to the small number of positive test results, the findings on risk factors should be interpreted with caution. Based on the serogroups found in the three species, dogs could be a source of infection for humans, or dogs and humans are exposed to the same environmental risk factors Clinical leptospirosis in humans and domestic animals should be investigated by testing blood and urine for the presence of leptospires by molecular methods (qPCR).