Questionnaire survey to assess knowledge and attitude of trypanocidal drug utilization practices, entomological survey to know apparent cyclical vector density and other mechanical vectors responsible for trypanosome infection transmission and parasitological study to elucidate the prevalence of bovine trypanosome was conducted in tsetse suppression areas and tsetse non-suppression areas of South Omo Zone, Southern Ethiopia from November 2018- May 2019. SPSS version 20 and R- software packages were used to analyze collected field data. For questionnaire survey, 124 cattle owners from tsetse suppression areas and 60 cattle owners from tsetse non-suppression areas were interviewed about their knowledge, attitude and practice (KAP) of trypanocidal drug utilization and constraints of trypanosome infection in the area. Blood samples from 1284 local breed cattle; 642 samples during dry season (344 from tsetse suppression areas and 298 from tsetse non-suppression areas) and 642 during wet season (344 from tsetse suppression areas and 298 from tsetse non-suppression areas) were parasitologically examined by using buffy coat technique and thin blood smear method. For entomological survey, 96 NGU traps were deployed (64 traps in tsetse suppression areas, 32 traps in tsetse non-suppression areas) in suspected Glossina multiplication habitats. The overall prevalence of bovine trypanosome infection in South Omo zone was found to be 11.05% (142/1284). The overall seasonal prevalence of bovine trypanosome infection in South Omo zone was 14.33% (92/642) and 7.78% (50/642) for dry and wet seasons, respectively. There was a statistically significant difference (P