The functionalization of cellulose fibre by grafting L-cysteine on the surface (Cys-fibre) significantly improves the sorption capacity toward mercury and methyl-mercury. The Cys-fibre is used to pack a mini-column for on-line separation and preconcentration of mercury species in a sequential injection system. The retained mercury and methyl-mercury could readily be recovered by a mixture of L-cysteine and nitric acid. The inorganic mercury is selectively quantified using the cold atomization mode with vapor generation atomic fluorescence spectrometry. The total amount of mercury is determined by adopting the flame/heat atomization mode, and thus the concentration of methyl-mercury is achieved by the difference. When 1000 μL of sample solution is processed followed by elution with 100 μL of eluent, detection limits of 1 ng L⁻¹ (3σ, n = 9) for inorganic mercury and 3 ng L⁻¹ (3σ, n = 9) for methyl-mercury are obtained within linear ranges of 0.01–0.7 μg L⁻¹Hg²⁺ and 0.03–2.0 μg L⁻¹MeHg, respectively. The precisions for Hg²⁺ and MeHg of 1.5% RSD (n = 11) and 2.6% RSD (n = 9) are recorded at 0.1 μg L⁻¹ and 1.0 μg L⁻¹, respectively. The procedure has been validated by analyzing mercury in certified reference material of BCR176 and a cosmetic sample. Speciation of mercury and methyl-mercury in a series of water samples and the extracts of cosmetic and seaweed samples are performed, and favorable spiking recoveries have been demonstrated.