The application of phenylfluorone (PF)-Mo(VI) complex as a spectroscopic probe is studied. In the presence of OP microemulsion at pH 3.04, PF-Mo(Ⅵ) complex combines protein rapidly to form a stable compound and the absorbance at 527 nm is in proportion to the concentration of protein in the range 0-16 μg mL-1 for bovine serum albumin (BSA). OP microemuslion media is introduced into protein determination, it has increased markedly the sensitivity of the system. The molar absorption coefficient was 5.98×l06 L mol-1 cm-1 for BSA. The assay, with sensitivity, simplicity and tolerance to many foreign substances, is applied to the determination of protein in samples with satisfactory results. Moreover, the binding number of BSA with the complex, which is determined by molar ratio and slope ratio methods, is in good agreement. The application of phenylfluorone (PF) -Mo (VI) complex as a spectroscopic probe is studied. The presence of OP microemulsion at pH 3.04, PF-Mo (VI) complex combines protein rapidly to form a stable compound and the absorbance at 527 nm is in proportion to the concentration of protein in the range 0-16 μg mL-1 for bovine serum albumin (BSA). OP microemuslion media is introduced into protein determination, it has increased markedly the sensitivity of the system. was 5.98 × l06 L mol-1 cm-1 for BSA. The assay, with sensitivity, simplicity and tolerance to many foreign substances, is applied to the determination of protein in samples with satisfactory results. Moreover, the binding number of BSA with the complex, which is determined by molar ratio and slope ratio methods, is in good agreement.