This study aimed to develop e-learning for pre-service science teachers and investigate the effect of this e-learning on their attitudes toward learning and teaching. A mixed method was employed in this research. Participants were 22 pre-service science teachers at one public university in Thailand who enrolled in the Educational Psychology and Guidance for Teacher class in the first semester of 2020. The instruments used in this study were a questionnaire with 20 items to investigate participants' knowledge, understanding, and opinions toward the e-learning lessons. The data analysis by mean, standard deviation, relative gain score, and content analysis. The results indicated that the developed e-learning effectively increases pre-service science teachers' attitudes toward teaching and learning. The relative gain score of attitudes toward teaching and learning was 89.47, and the high satisfaction level towards e-learning (mean = 3.87). The findings show that participants reflected that e-learning lessons helped learners manage learning independently, learn from any place, and repeat learning to learn anytime. However, feedback highlighted areas for improvement, including the need for more engaging audio narration and detailed explanations in exercises of this e-learning because it was monotonous.