Knowledge about One Health approach and its importance among the practitioners and the community in addressing health threats is of paramount important. This study aimed to assess knowledge of One Health actors on multi-sectoral coordination in addressing health risks in selected regions of Tanzania. A descriptive study was conducted from June to August 2023 in selected regions of Arusha, Iringa and Dodoma where quantitative and qualitative data were collected from 101 participants representing the line ministries and sub-national regional and district levels. Descriptive analysis was done using EPI INFO 7.2.5.0 statistical software and Microsoft excel. Results showed a decreasing proportion of participants reporting to be aware of One Health approach at the National level with 100.0% awareness and only 32.3% awareness at the district level. It was also revealed that 76.9% of the participants at the national level had received One Health training through short courses, workshops meetings, and Continuous Professional Development (CPD) courses compared to the varying proportions of 57.7% and 19.4% at the regional and district level, respectively. Limited awareness of different professional disciplines on One Health approach is the major challenge in its operationalisation and institutionalisation at sub-national levels. Awareness creation to One Health actors and advocacy to policy and decision-makers at all levels will enhance implementation, multi-sectoral coordination and collaboration among key actors in addressing health risks emergencies.