Through a questionnaire survey, this study aims to investigate the contributions of different sources in developing science teachers’ practical knowledge of teaching with practical work. The framework of this study is based on the conceptualization of PCK and ten possible sources of science teachers’ PCK related to teaching with practical work. The findings of this study can be summed in the following aspects. Firstly, among the ten possible sources, overall, the more important sources are “personal teaching practices and reflection” and “informal exchanges with colleagues”; the important sources are “reading teaching materials,” “learning from online resources,” and “pre-service training experience,” while the least important ones are “in-service training experience” and “experience as primary and secondary school student.” Secondly, for the five components of PCK of teaching with practical work, the contributions of the ten sources are different. Thirdly, three sources are found to be significantly different between teachers who have pre-service training experience and those who do not have. They are “classroom observation” (goals toward practical work), “in-service training experience” (laboratorial curriculum materials; instructional strategies), and “reading professional journals” (teaching assessment). In the last part of this paper, some explanations are provided for these findings and the limitation of this study is discussed.