Aim: In the last decade many survey studies have analyzed factors that influenced e-learning such as computer skills, access to the Internet and attitudes towards e-learning. Some surveys were developed for broader studies and different contents while others lack psychometric properties testing. The aim of this study was to construct and examine the psychometric properties of a questionnaire measuring students’ attitudes towards e-learning. Methods: Research was conducted from October 2010 to October 2011. Participants were 308 undergraduate students (106 men and 202 women) from the School of Medicine and the School of Engineering at the University of Rijeka, Croatia. In the first version of the questionnaire, we generated 40 statements (items). Twenty-two statements were based on relevant scientific literature, whereas the remaining 18 statements were developed on the basis of our own knowledge and experience. Two experts in questionnaire construction and validation and two experts in e-learning reviewed independently all the statements and removed those with similar or ambiguous meaning. The remaining 24 statements were chosen for the validation. Factor analysis was used to find the factor structure of the questionnaire and to measure construct validity. Results: The final version of the questionnaire consisted of 22 items. The Principal Component Analysis (PCA) determined two factors: factor I – positive attitudes towards e-learning (10 items) and factor II – negative attitudes towards e-learning (12 items). Correlation between factors was 0.67. The reliability of the questionnaire was high (a=0.87). Participants had positive attitudes towards e-learning with a mean score ± standard deviation of 87±10 out of maximum 110. Conclusion: The instrument proved to have good psychometric properties and it can be used for future studies assessing attitudes towards e-learning.