To obtain real-time dynamic changes of non-protein-bound copper in the blood, we have developed an online microdialysis sampling system coupled with a flow-injection graphite furnace-atomic absorption spectrometer (FI-GFAAS). The analytical performances of the online system such as linearity, limit of detection, precision, and spiked recoveries were validated. Before the in vivo experiments, the in vivo recovery was conducted. The levels of non-protein-bound Cu in the blood of living rabbits were evaluated before and after administering them with 5 mg/kg body weight of CuSO4 by the online microdialysis-FI-GFAAS system. The results showed that the avarage basal concentration of non-protein-bound Cu in the blood of living rabbits was 16.2 μg/L (n=3). Furthermore, the levels of non-protein-bound Cu in the blood of living rabbits were observed after a long delay following intravenous injection of CuSO4. The non-protein-bound Cu reached the maximum value at 125 min after injection. Our present study might provide the in vivo, direct observation that different metals have their own binding characteristics with proteins when transported into the blood of living organisms.