Inquiry teaching in science education has been widely advocated for decades. It is a critical learning objective in many science teacher preparation programs. Despite its importance, it is not effectively implemented in science classrooms. One of the reasons is the lack of reliable and valid instruments that provide practical definition, concrete guideline, and objective assessment for the practice of inquiry teaching. To fill this gap, we designed a 3E rubric based on the 5E learning model as one specific form of inquiry teaching to measure preservice teachers’ practice at different phases of a science lesson. In this study, we thoroughly introduced the 3E rubric and its use. Through drawing on 76 elementary pre-service science teachers’ teaching videos, we analyzed its reliability and validity with the tools of Intraclass Correlation Coefficient, Fleiss’ kappa, and Pearson correlation. According to the results, the 3E rubric is a reliable and valid tool to assess pre-service teachers’ practical knowledge of inquiry teaching. The contributions of this rubric to the teaching and research in science teacher preparation are discussed and future research directions are proposed.