Abstract Background Continuing professional development (CPD) is required for health workers in practice to update knowledge and skills regularly to match the changing complexity of healthcare needs. The objective of this study was to identify the training needs of Medical Laboratory professionals in Ethiopia. Methods A total of 457 medical laboratory professionals from five regions and two city administrations were involved in the study. Data were collected from August 02 to 21, 2021 with structured self-administered online tool with five-point Likert scale. The tool had consent, demography, cross-cutting issues, and main activity area specific to medical laboratory. Results Majority of the participants were male (80.1%). Participants from Amhara region 110 (24.1%) were the largest groups in the survey followed by Oromia 105 (23%) and Addis Ababa 101 (22.1%). The study participants comprised 54.7% with a bachelor’s degree, 31.3% with a diploma (associate degree), and 14% with a master’s degree. The participants had varying years of service, ranging from less than one year to over 10 years of experience. Most of the participants work as generalists (24.1%) followed by working in microbiology (17.5%) and parasitology (16%). The majority (96.9%) were working in a public sector or training institutions and the rest were employed in the private sector. Our study showed that the three most important topics selected for training in the cross-cutting health issues were health and emerging technology, computer skills and medico-legal issues. Topics under microbiology, clinical chemistry and molecular diagnostics were selected as the most preferred technical areas for training. Participants have also selected priority topics under research skill and pathophysiology. When the laboratory specific issues were regrouped based on areas of application as technical competence, research skill and pathophysiology, thirteen topics under technical competence, four topics under research skill and three topics under pathophysiology were picked as priority areas. Conclusion In conclusion, our study identified that CPD programs should focus on topics that address technical competence in microbiology, clinical chemistry and molecular diagnostics. Additionally competencies in research skill and updating knowledge in pathophysiology should also receive due attention in designing trainings.