Aedes aegypti is a major vector of Dengue fever, whose slight reappearance was recorded in the Red Sea Governorate in 2017. The present study was carried out to determine the DNA barcoding of Ae. aegypti and investigate the sensitivity of its aquatic immature stages to different insecticides, with reference to non-target organisms, zebrafish, Danio rerio, and Daphnia magna. Genetic identification of Ae. aegypti was confirmed through PCR amplification and subsequent sequencing. The obtained DNA sequences were deposited in the National Center for Biotechnology Information (NCBI) sequence database with the accession number OR76138. Methomyl, α-cypermethrin, and chlorpyrifos were tested against immature stages of Ae. aegypti. Additionally, the effect of tested insecticides on acetylcholinesterase (AChE) and glutathione S-transferase (GST) activities were assessed. Results showed that the activity of the tested insecticides varied according to the concentration of the insecticides, exposure time and the immature stage tested. Complete larval mortality (100%) of the first instar was attained at 0.007, 0.0009, and 0.005ppm of methomyl, α- cypermethrin, and chlorpyrifos after 48h of exposure, respectively. Generally, α- cypermethrin was the most effective insecticide, followed by chlorpyrifos and methomyl. In addition, methomyl caused the least inhibition of AChE activity (5.53 and 5.90U/ L) in larvae I after 24 and 48h of exposure, compared to 5.74 and 6.13U/ L for the control group. In addition, the GST activity was significantly increased (P< 0.05) by all tested insecticides. The highest GST values (1.73 and 1.59U/ g tissue) were recorded for pupae after 24 and 48h of exposure to α-cypermethrin. On the other hand, methomyl recorded LC₅₀ of 0.862, 0.011, and 0.575, 0.009 ppm against zebrafish and Daphnia after 24 and 48h, respectively. Generally, methomyl, α- cypermethrin, and chlorpyrifos are effective agents against Ae. aegypti, however, their environmental footprint cannot be overlooked.