BackgroundIn the Somali region of Ethiopia, visceral leishmaniasis (VL) is a public health concern. However, VL epidemiology and sand fly vectors have not been well studied in various areas of the regional state, including Denan district. Therefore, this study was conducted to determine the sero-prevalence, associated factors, and distribution of sand fly vectors of VL in Denan district, south-eastern Ethiopia.MethodsA facility-based cross-sectional study was conducted from April to September 2021 among VL patients with classic signs and symptoms visiting Denan Health Center in south-eastern Ethiopia. Using a convenience sampling method, 187 blood samples were collected from individuals who visited Denan Health Center during the study period. Blood samples were subjected to Direct Agglutination Test for the detection of antibodies to VL. A pre-tested structured questionnaire was also used to gather information on risk factors and other characteristics of knowledge and attitude assessment. Sand flies were also collected from indoor, peri-domestic, mixed forest, and termite mounds using light and sticky traps to determine the fauna and abundance.ResultsThe overall sero-prevalence rate was 9.63% (18/187). The sero-prevalence was significantly associated with outdoor sleeping (OR = 2.82), the presence of damp floors (OR = 7.76), and sleeping outdoor near animals (OR = 3.22). Around 53.48% of the study participants had previously heard about VL. Study participants practiced different VL control methods, including bed nets (42%), insecticide spraying (32%), smoking plant parts (14%), and environmental cleaning (8%). In total, 823 sand fly specimens, comprising 12 species in two genera (PhlebotomusandSergentomyia), were trapped and identified. The most abundant species wasSergentomyia clydei(50.18%), followed byPhlebotomus orientalis(11.42%). Also, a higher proportion ofP.orientaliswas found in termite mounds (65.43%), followed by mixed forest (37.8%) and peri-domestic (20.83%) habitats.ConclusionThe study demonstrated a 9.63% sero-positivity of VL and a remarkable gap in knowledge, attitude, and practices towards VL.P. orientaliswas also detected, which could be a probable vector in this area. Thus, public education should be prioritized to improve the community’s awareness of VL and its public health impact. In addition, detailed epidemiological and entomological studies are recommended.