The cotton leaf worm, Spodoptera littoralis (Boisd.) is a key pest of cotton and a wide range of economically important crops, vegetables, and fruits in Egypt and worldwide. Emamectin benzoate is a broad-spectrum bioinsecticide that has long been used to control lepidopteran pests such as S. littoralis. The toxicity of emamectin benzoate against S. littoralis fourth-instar larvae and its biochemical effects on their acetylcholinesterase (AChE), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were determined. The toxicityof emamectin benzoate against larvae of S. littoralis was increased by the increase of exposure time. The LC₅₀ values (lethal concentrations causing 50% larval mortality) caused by emamectin benzoate against S. littoralis larvae were 8.8×10^-2, 1.1×10^-2 and 4.8×10^-3 ppm a.i. at 2, 3 and 4 DAT, respectively. Overall, the application of emamectin benzoate on larvae at concentrations of 4.8×10^-4, 9.6×10^-4 and 2.4×10^-3 ppm a.i. (concentrations equivalent to 10, 20 and 50% of the estimated LC₅₀ value at 4 DAT, respectively) increased the activity of AChE, AST, ALT of the treated larvae. The increase rate in the activity of those enzymes was dependent on both the concentration and exposure time for emamectin benzoate.