The prospective teachers can effectively master Technological Pedagogical and Content Knowledge (TPACK) through the application of technology-based guided inquiry as an effort to enhance technology literacy, learning quality and also as a medium for problem-solving. This study aimed at analyzing the impact on the implementation of technology-based guided inquiry to improve students' TPACK ability. This study employed a one-group pretest-posttest design. The research population was the students of Biology Education Department, Syiah Kuala University, Indonesia. The research sample was 47 students taken with purposive sampling technique. The research instruments employed a test and a questionnaire. The data were analyzed using a paired sample t-test and quantitative descriptive. The results showed that students' understanding of TPACK according to all assessment aspects can be categorized as "very good." In details, the content knowledge had a higher posttest score than the pretest. Therefore, it can be concluded that the application of technology-based guided inquiry has a significant impact to improve the students' understanding of TPACK including Content Knowledge (CK), Pedagogical Knowledge (PK) and Technological Knowledge (TK). The application of guided inquiry learning on TPACK can restructure teachers' experience for their professional development by designing their own learning materials with technology use.