The aim of the study is to explain the effect of portfolio use on the development of critical thinking strategies of pre-service teachers with different learning approaches. The study used a case study design. The sample consisted of 26 pre-service biology teachers. The Study Process Questionnaire developed by Biggs was used to determine the learning approaches of pre-service teachers. Data were collected through interviews and portfolios. The data obtained were analyzed by descriptive analysis method. Through the use of portfolios, it was found that pre-service teachers improved in questioning what they learned, using time and finding solutions. It was also found that critical thinking skills of pre-service teachers with surface learning approach were at a lower level. It was determined that pre-service teachers with strategic and deep learning approaches differed from pre-service teachers with surface learning approach in terms of establishing relationships between the subjects they learned. The skills of finding a solution to the problem encountered, which is one of the critical thinking skills of pre-service teachers with deep and strategic learning approach, was developed through the portfolio. It is recommended that teachers should develop instructions and activities suitable for each learning approach by considering learning approaches in developing critical thinking.