<p>Critical thinking is an important component of scientific literacy. Understanding the actual situation of students’ critical thinking can help science teachers improve their teaching methods. The purpose of this study was to construct a theoretical framework and develop a valid and reliable assessment questionnaire to evaluate middle school students’ critical thinking in science classrooms. First, under the guidance of Paul’s critical thinking theory, this study initially constructed a theoretical framework for critical thinking in science classrooms. It then aimed to empirically evaluate the level of consensus on the framework through a three-stage Delphi questionnaire, involving 37 participants selected from renowned communities of leading experts, including scientists, science educators, and philosophers of science. Second, based on the theoretical framework, this study developed a corresponding assessment questionnaire and provided evidence for the validity and reliability, resulting in a set of two first-level dimensions of critical thinking dispositions and abilities with several second-level indicators, respectively. Finally, this study discussed how to utilize the assessment questionnaire to middle school students in science classrooms.</p>