Background: In Uganda, there are persistent weaknesses in obtaining accurate, reliable and complete data on local and external investments in immunization to guide planning, financing, and resource mobilization. This study aimed to measure and describe the financial envelope for immunization from 2012 to 2016 and analyze expenditures at sub-national level.Methods: The Systems of Health Accounts (SHA) 2011 methodology was used to quantify and map the resource envelope for immunization. Data was collected at national and sub-national level from public and external sources of immunization through key informant interviews coupled with document reviews. Data were coded using the SHA, categorized and disaggregated to detail the expenditure on immunization activities. Results: Over a five-year period, funding for immunization increased fourfold to US$ 85.6 million in 2016. The Ugandan government was the main contributor (55%) to immunization resources from 2012 to 2014. However, Gavi, the Vaccine Alliance contributed the majority (59%) of the resources to immunization in 2015 and 2016. Majority (66%) of the funds were managed by the National Medical Stores. Over the five-year period, 80% of the funds allocated to immunization activities were spent on facility based routine immunization (expenditure on human resources and outreaches). At sub-national level, districts allocated 15% of their total annual resources to immunization to support supervision to lower health centers (including distribution of vaccines). Health facilities spent 5.5% of their total annual resources on immunization to support outreaches.Conclusion: Development partner support has aided the improvement of vaccine coverage and increased access to vaccines. However, there is an increasing dependence on this support for a critical national program raising sustainability concerns alongside other challenges like being off budget and unpredictable. To ensure financial sustainability, there is need to operationalize the immunization fund, advocate and mobilize additional resources for immunization from the Government of Uganda and the private sector, increase the reliability of resources for immunization as well as leverage on health financing reforms like the National Health Insurance.