The aim of this study was to construct a criterion-based reference test for the human body unit in the sixth-grade science subject, using the Rasch model of Item Response Theory. The study aimed to determine the appropriate cut-off score for the test, and to verify its psychometric properties in classifying students into proficient and non-proficient levels. The achievement test, consisting of 50 items, was administered to a sample of 1003 male and female students. The researcher employed the ItemMapping method after verifying the assumptions of the Rasch model and excluding individuals who did not meet these assumptions, in order to calculate the cut-off score. The person-item map of individuals and items was used to verify the validity of test questions for different ability levels. Additionally, the Rudner method was used to calculate the accuracy and consistency of student classification, as well as the overall alpha and beta reliability coefficients. Furthermore, the person and item separation indices were calculated using the winsteps software. The results indicated good validity and reliability of the test, with a classification accuracy and consistency of 0.93 and 0.90, respectively. The overall alpha and beta reliability coefficients were 0.78 and 0.74, respectively, and the person separation index was 2.87, while the item separation index was 10.65. Differential item functioning was also examined using the MantelHaenszel method based on gender. The results revealed several items with differential functioning in favor of both males and females. Among these items, seven had moderate differential functioning, while only one item had high differential functioning. Based on the current study results, it is recommended to utilize the criterion-reference test for diagnosing students as proficient or non-proficient and rely on these results in designing remedial and enrichment plans. Moreover, several suggestions for future research were provided based on the findings of the current study.