Developing students' scientific literacy is the most important educational goal and challenge of the 21st century. Many studies have confirmed that flipped learning has significantly impacted learning science. Researchers indicate that the lack of an appropriate learning guidance strategy in the pre-class stage for flipped learning will influence students' understanding of learning content and affect in-class learning activities. In order to tackle this problem, the present study proposed a flipped learning approach based on identification, summarization, self-reflective questioning, and application (ISSA), further exploring the influences on students' scientific literacy, communication tendency, problem-solving tendency, learning motivation, and cognitive load. In addition, the study used a true experimental design to assess the effectiveness of the proposed learning method, and 58 university students were recruited to participate in a natural science course. The experimental group (N = 29) adopted the proposed learning approach, while the control group adopted the conventional flipped learning approach. The results showed that the experimental group had higher scientific literacy, communication tendency, problem-solving tendency, and extrinsic motivation than the control group. The interviews showed that the ISSA flipped learning method could improve students' understanding of the learning content. In particular, the process of peer interaction promoted their self-reflection and scientific literacy skills.