Indonesia is an archipelago with a tropical climate. This condition facilitates the spread of dengue fever, causing infections every year. Based on the Bali Provincial Health Profile has seen an upward trend in the Incidence Rate (IR) of dengue fever, with rates of 22.4 (2018), 137.3 (2019), and 278.6 (2020) per 100.000 population so that in 2020, Bali contributed the highest IR when compared to other provinces in Indonesia. Cases of vector resistance to synthetic larvicides have also been reported in various previous studies. The purpose of this study is to analyze the potential of ethanol extract of Tulsi plant (Ocimum tenuiflorum) as a natural larvicide on Aedes aegypti instar III larvae. This research involved creating Tulsi extract using the maceration method and testing its larvicidal activity against A. aegypti larvae in a laboratory setting. Extract was obtained by maceration method using 96% ethanol solvent and tested at concentrations of 0.03%, 0.06%, 0.12%, 0.24%, and 0.48%. Each concentration underwent three replicates over 24 hours. The results showed that as the concentration of Tulsi extract increased, there was a significant increase in the death of A. aegypti larvae with significant differences based on the Kruskal Wallis test (P value < 0.05). The 50% lethal concentration value (LC50) was determined to be 0.135%, while the 99% lethal concentration value (LC99) was determined to be 0.25%. This research succeeded in proving the potential of Tulsi extract as a larvicide made from natural ingredients. Further research is needed to be able to create practical alternative larvicide formulations based on Tulsi.