<p>Nitrogen isotope fractionation (ε¹⁵N) in sedimentary rocks has provided evidence for biological nitrogen fixation, and thus primary productivity, on the early Earth. However, the extent to which molecular evolution has influenced the isotopic signatures of nitrogenase, the enzyme that catalyzes the conversion of atmospheric nitrogen (N₂) to bioavailable ammonia, remains unresolved. Here, we reconstruct and experimentally characterize a library of synthetic ancestral nitrogenase genes, spanning over 2 billion years of evolutionary history. We assess the resulting ε¹⁵N values under controlled laboratory conditions. All engineered strains exhibit ε¹⁵N values within a narrow range comparable to that of modern microbes, suggesting that molybdenum (Mo)-dependent nitrogenase has been largely invariant throughout evolutionary time since the origins of this pathway. The results of this study support the early origin of molybdenum nitrogenase and the resilience of nitrogen-isotope biosignatures in ancient rocks, while also demonstrating their potential as powerful tools in the search for life beyond Earth.</p>