In this study, a 'biotechnology literacy test' was developed to determine the biotechnology literacy of prospective science teachers, and its validity and reliability were determined. For this purpose, 42 items were prepared by considering Bybee's scientific literacy classifications (nominal, functional, procedural, and multidimensional). The experts were asked to rate each item for content validity. As a result of the experts' evaluations, the content validity of the test was confirmed. The draft test was applied to 494 science prospective teachers. Difficulty and discrimination coefficients of items within the test were calculated to provide evidence of construct validity. It was decided to exclude 13 items as a result of item analysis. It can be said that that the test was difficult (p = 0.40) and discriminative (D = 0.41) at medium level. Finally, a valid and reliable "biotechnology literacy test" consisting of 25 multiple-choice items each with five choices was developed in this research. (c) 2019 International Union of Biochemistry and Molecular Biology, 47(2): 179-188, 2019.