The objectives of this study was to determine the effect of lemon juice (Citrus Limon (L.) Osbeck) as larvae of Aedes aegypti in third instar, and to determine the difference in mortality of Aedes aegypti larvae in third instar by giving different concentration of lemon juice (Citrus Limon (L.) Osbeck). This study is belonging to experimental research. The sample in this study was lemon juice with 5 times treatment, namely concentration 5%, 10%, 15%, 20%, and 25% which calculated with the replication formula. According to the finding of the research shows that in the of 10% concentration was effective in killing all Aedes aegypti larvae due to the average larval mortality of 100%, and the 15%, 20%, and 25% concentration of obtained larval mortality results of 100%. The data analyzed by using One Way ANOVA showed that probability/sig value was <0.05, therefore, H0 is rejected, while the result of f count 103,143> f table. 2.866 was obtained. Result f count> f table then H0 is rejected and H1 is accepted. It can be concluded that there was an effect of lemon juice (Citrus Limon (L.) Osbeck) as larvae of Aedes aegypti in third instar.