Wereport the first comprehensive insecticide susceptibility status of Aedes aegypti (L.) larvae from Singapore. The study indicated that Ae. aegypti is susceptible to temephos, although resistance (RR50 =1.29–4.43-fold) could be developing. Of high concern is the detection of moderate to high resistance to permethrin (RR50 = 29–47-fold) and etofenprox (RR50 = 14–34-fold). Biolarvicide Bacillus thuringiensis israelensis (Bti) remains effective. The insecticide susceptibility profile of Ae. aegypti larvae was found to be homogenous among the different sites studied across the island city. The addition of synergists piperonyl butoxide, S,S,S,-tributyl phosphorotrithioate, and triphenyl phosphate generally failed to enhance the toxicity of the insecticides investigated, suggesting an insignificant role of metabolic-based resistance, and a possible involvement of target site resistance. Further biochemical investigation of specific metabolic enzyme activities suggested that detoxifying enzymes, mono-oxygenases, esterases, glutathione S-transferases, and altered acetylcholinesterases, generally did not contribute to the resistance observed. This study clearly demonstrated that pyrethroid resistance is widespread among Ae. aegypti population and lowered susceptibility to organophosphates is developing.