Systems thinking (ST) is an essential competence for future life and biology learning. Appropriate assessment is critical for collecting sufficient information to develop ST in biology education. This research offers an ST framework based on a comprehensive understanding of biological systems, encompassing four skills across three complexity levels. Employing a construct-modelling approach, this study developed and validated a Biological Systems Thinking Test (BSTT) grounded in the systems view of life, aimed at middle school students. The BSTT was administered to 456 seventh and eighth graders in China. Partial credit model analysis indicated that the BSTT possesses satisfactory reliability and validity. The Martin-Löf test and the likelihood-ratio test results confirmed that the BSTT provides precise measures of ST in biology when the skills are considered a unified construct. Furthermore, the Wright map demonstrated the comparability of student ability distributions and item difficulty. The increased mean difficulty thresholds of item responses aligned with the three performance levels, and the distinguishable three-level distribution in the Wright map confirmed that the BSTT effectively encompasses students' ST levels. The BSTT offers teachers and educators a valuable assessment tool for evaluating students' ST performance in biology.