Continuous use of chemical larvicides causes resistance in many species around the world and also environmental pollution. Nanoemulsions of herbal larvicides (such as essential oil based larvicides) are suitable alternatives for this purpose. However, no work so far has investigated the stability of a nanoemulsion after 100–200 times dilution and its effect on larvae of Anopheles stephensi, a major vector spreading malaria. Tarragon is a common medicinal plants and its essential oil had effective larvicidal activity. Different nanoemulsions of Tarragon essential oil were prepared using various amounts of surfactants and co-surfactants. For the first time, a comparison of larvicidal activity between two optimized preparations (particle sizes of 15.6 & 14.5 nm) with tarragon essential oil against Anopheles stephensi was performed. The components of Tarragon essential oil were identified by GC–MS analysis. Forty eight components were determined, with 5 major components including P-Ally anisole (67.623%), cis-Ocimene (8.691%), beta-Ocimene Y (7.577%), Limonene (4.338%) & 3-Methoxy cinnam aldehyde (1.49%). The bioassay of essential oil was performed, LC at 50 or 90% with values of 11.36 or 17.54 ppm, were calculated using probit analysis. No larvicidal effect was observed from nanoemulsion inactive ingredients (i.e. Tween 20 and isopropyl alcohol). Larvicidal activity of the most stable formulation after dilution (particle size 14.5 nm), showed significant increase compared to bulk of the oil and nanoemulsions with lesser stability profile after dilution. By designing and preparing a stable essential oil nanoformulation, against dilution, which takes the advantage of lesser volatile properties. And also, achieving to effective and environmentally friendly larvicides with low cost will be possible.