Background In-service training programs should be evaluated and modified regularly to provide quality health services. However, in Ethiopia, there is no published evidence about its effectiveness. Therefore, we evaluated the effectiveness of in-service training program of Amhara Public Health Institute Dessie Branch (APHI) using the Kirkpatrick model. Methods In October 2019, a concurrent nested mixed quantitative/qualitative, facility-based cross-sectional study was conducted among 107 stakeholders from 22 randomly selected east Amhara government health facilities. The qualitative part also involved all this key stakeholder interviews. Data was collected using a semi-structured questionnaire through face to face interview. Epi data manager and Microsoft Excel 2016 software’s were used for data entry and analysis respectively. The major qualitative findings were narrated and summarized based on thematic areas to supplement the quantitative findings. Results A total of 107 laboratory professionals working in 22 health facilities were interviewed with a response rate of 97.3%. At reaction level, 82.1% of participants strongly agreed/agreed with the course structure, training contents and learning tools. Likewise, 85.4% of them strongly agreed/agreed on trainer's knowledge and their communication skills. Besides, 93.1% of them stated an improvement in knowledge and skills after attending training. Yet, only 65.6% of participants transferred their knowledge and skills in to practice. Regarding the training set ups and environment, 45.1% of the respondents strongly disagreed/disagreed with the training hall, toilet, café, financial process, accommodation perdiem and, tea & snacks. Conclusion and recommendations: Generally speaking, the laboratory in-service training program of APHI was more or less effective. Our finding suggests regular monitoring and evaluation of training events based on a standard set of performance indicators. Furthermore, the institute is mandated to create a conducive learning environment and well established training set ups for trainees.