By adding L-cysteine into the mobile phase of chromatography, a simple on-line high performance liquid chromatography-cold vapor generation-atomic fluorescence spectrometry (HPLC-CV-AFS) method for separation and determination of divalent mercury (Hg2+), monomethylmercury (MMC) and ethylmercury (EMC) was developed. Compared with the conventional HPLC-UV-CV-AFS hyphenated systems, the proposed HPLC-CV-AFS system is very simple and low cost as no strong oxidant, no additional post-column interface, such as ultraviolet or microwave, and no organic solvent were needed. Parameters influencing mercury separation and determination were optimized in detail. The detection limits for MMC, EMC and Hg2+, based on three folds of baseline noise and 100 μL injection, were 0.05, 0.07, and 0.1 μg L−1, respectively. The relative standard deviation (RSD) for three parallels determinations varied from 1.4 to 2.5% at a concentration level of 50 μg L−1. The proposed method was successfully applied to analyze two certified reference material, DORM-2 (dogfish muscle) and TORT-2 (lobster hepatopancreas), and biological samples.