Abstract: A method of complex-extracting-spectrophotometric determination of nicotine in an aqueous tobacco extract was developed by virtue of the principle that the light absorptivity of a complex of nicotine and methyl-orange is in proportion to its nicotine concentration while the complex is in chloroform. In this method, 2.0 mL tobacco extract was reacted with 2.0 mL saturated methyl-orange solution in the presence of 2.0 mL (pH5.0) citric acid/sodium hydrogen phosphate buffer solution, extracted with 20mL chloroform at room temperature for 2 to 3 minutes, stood for 3 to 5 minutes, and then determined the light absorptivity at the wavelength of 420 nm within 6 minutes. The content of nicotine was calculated with the calibration curve. The light absorptivity of the complex was linearly correlated with its nicotine concentration in the range of 0.0425-0.2550 mg/mL, correlation coefficient was 0.9994. The average recovery of nicotine was 98.32% and its CV was less than 5%.