2-thioxanthone thioacetic acid (TXSCH2COOH, T), which has a fluorometric character, was used for new fluorometric system upon Fe(II) analysis in biological samples as the main target. T-BSA binary complex was firstly consisted with non-covalent interactions between T and BSA at the equilibrium concentration as 1.77 10-4.M. T-BSA binary complex emission was increased at the ratio of 24.40% due to stabilization property of BSA (pH:7), compared with T emission intensity. Fluorescence emission spectroscopy was used for the all measurements because of an economic, a sensitive and a practical method compared with other spectroscopic analysis. T-BSA-Fe(II) triple complex was also obtained by adding Fe(II) ion to T-BSA binary complex solution. Its characterization was performed to be investigated with optimum excitation wavelength, buffer concentration, pH and temperature as 297 nm, 10-3 M Tris HCl (10-2M NaCI), pH:7.2 at 25 C, respectively. The results of Fe(II) analysis in serum showed a certain response in fluorometric T-BSA-Fe(II) triple complex measurement system as 50.42 ± 5.8 mg/dL. The analyses of our fluorometric triple complex system were compared with the reference electrochemiluminescence method and similar results were obtained. Fluorometric measurements of T-BSA-Fe(II) triple complex, its characterization and Fe(II) analysis in this system have not been investigated in literature gives originality to our study.