Extraction of L-lysine with sec-octylphenoxy acetic acid (CA-12) in sulfonated kerosene was investigated under different pH and CA-12 concentrations, and optimized conditions were determined. Half-saturation fluorometric analysis and atomic force microscopy showed that the electrovalent bond between L-lysine and CA-12 acted as the main force to partition L-lysine into organic phase. Moreover, the mathematically analyzed extraction process indicated that the extractive complex was a 1:2 L-lysine center dot CA-12 structure. The initial extraction efficiency could reach 78.9 +/- 1.2% and the total strip extraction efficiency after three strip extractions could reach 95.7 +/- 1.8% by using a dilute KCl solution.