Background Despite the widely acknowledged uses of technology-enhanced learning in medical education, the evaluation approach for these online learning environments specific to medical education are limited. Identifying online learning environment key components for medical education may provide an opportunity to address this gap. This study aimed to develop and validate Digital Medical Education Environment (Digi-MEE) Instrument that measures the online learning environment in medical education.Methods Questionnaire development and validation studies were conducted involving 726 participants in total. Components and items of online learning environments were generated and identified through a scoping review and a modified e-Delphi study. A panel of selected content experts carried out content validation leading to calculation of content validity index (CVI). A group of medical students performed face validation leading to determination of face validity index (FVI).This validation process was followed by exploratory factor analysis, confirmatory factor analysis and assessment of reliability to determine Digi-MEE’s factorial structure and internal consistency.Results A total of 15 experts reviewed nine components with 73 items of Digi-MEE to appraise its content validity. As a result, 26 items were removed, and 17 items were revised that led to Digi-MEE 2.0 with 47 items. The S-CVI/Ave was 0.98. Following response process validation, the number of items were reduced to 46, S-FVI/Ave of the Digi-MEE 3.0 was 0.87. Exploratory factor analysis yielded 46 items within nine components and depicted 57.18% variance. After confirmatory factor analysis, the final version of Digi-MEE contained 28 items within nine components. All goodness of fit indices achieved acceptable level. The overall Cronbach alpha of the final Digi-MEE was more than 0.90, while for the nine constructs ranged in between 0.62 and 0.76.Conclusion This study proposed a promising valid and reliable instrument, Digi-MEE, to evaluate online education environment in medical education. The content, response process, factorial structure and internal consistency evidence support the validity of Digi-MEE. Medical schools can use Digi-MEE as an evaluation tool for the continuous quality improvement of online learning environments.