So far, instruments to measure genetic literacy that encompass global genetic issues are limited. In addition, instruments that assess practical knowledge and comprehensively evaluate genetic literacy skills have yet to be developed. Therefore, this research aimed to develop, validate, and improve an instrument based on a new conceptual framework of genetic literacy across grades. Six stages were conducted based on construct modeling to establish the Genetics Literacy and Reasoning Test. In the development process, numerous sources of evidence were collected, including evaluation from an expert panel to examine face validity, a pilot study to ensure comprehension, and testing involving 250 pre-service biology teachers. The results showed that the Genetics Literacy and Reasoning Test generally has good quality regarding dimensionality, reliability and separation, item fit, person-item mapping, and other data collected from interviews. This study highlights the importance of developing reliable and valid instruments to evaluate students' genetic literacy and recommends it as a valuable tool for research and practice.