Resistance to insecticides mainly occurs due to changes in insect metabolic enzyme. Increased metabolism is often caused by qualitative or quantitative changes of esterase and glutathione S-transferase. Susceptibility test and biochemical assay to detect organophosphate and synthetic pyrethroid resistance were conducted on Aedes aegypti from Wonosobo (new highland Dengue endemic area). The test were performed on F1 generation of Ae.aegypti field caught mosquitoes which aimed to determine the resistance mechanisms regarding two detoxifying enzymes i.e. esterase and glutathione S-transferase. Susceptibility test showed 23.4 and 46.7% mortalities after exposure to 0.8% malathion and 0.05% cypermethrin. The biochemical assay result suggested that esterase, monooxygenase and glutathione s-transferase activity tend to increase in Ae.aegypti in Wonosobo. Interview and questionaires conclude that synthethic pyrethroid was the only insecticide type used in vector control program by Wonosobo Health Office and was the most frequent insecticide type to be used in household by Wonosobo society to control Ae.aegypti population. Knowledge of localized resistance and underlying mechanisms helps in making rational decisions in selection of appropriate and effective insecticides in the event of a dengue outbreak.