The application of 5’-nitrosalicylfluorone (5’-NSF)-molybdenum(VI) complex as a spectroscopic probe was studied. In the buffer medium of HOAc-NaOAc at 3.45 and in the presence of PVA 124 microemulsion, 5’-NSF-Mo(VI) complex combines protein rapidly to form a stable compound, leading to an absorbance decrease at 525 nm of 5’-NSF-Mo(VI) complex. According to this change, microdetermination of protein has been described. Bovine serum albumin (BSA) could be determined in the linear range of 0—16 g昺L-1 with the detection limit of 11 ngmL-1. Many amino acid and metal ions studied do not interfere with the assay. The method possesses high sensitivity as well as high selectivity. It can be used to determine protein in human urine and milk powder successfully. The relative standard deviations are in all instances less than 4.7%, and the recoveries are between 97.6% and 106.0%. Moreover, the binding number of BSA with the complex, which is determined by using molar ratio, Rosenthanl graphic and slope ratio methods, is in good agreement with each other. The application of 5’-nitrosalicylfluorone (5’-NSF) -molybdenum (VI) complex as a spectroscopic probe was studied. In the buffer medium of HOAc-NaOAc at 3.45 and in the presence of PVA 124 microemulsion, 5’-NSF- According to this change, a microdetermination of protein has been described. Bovine serum albumin (R) (TM) BSA) could be determined in the linear range of 0-16 g 昺 L-1 with the detection limit of 11 ngmL-1. Many amino acid and metal ions studied do not interfere with the assay. The method possesses high sensitivity as well as The relative standard deviations are in all instances less than 4.7%, and the recoveries are between 97.6% and 106.0%. Moreover, the binding number of BSA with the complex, which is determined by using molar ratio, Rosenthanl graphic and slope ratio methods, is in good agreement with each other.