A novel, simple, sensitive and selective spectrofluorimetric method has been developed for the assessment of Carbohydrate Antigen (CA 15.3) in serum samples of breast cancer patients by using photo probe Tb-Bipyridyl (Tb-Bipy) complex. The determination method Based on the quenching of the luminescence intensity of Tb-Bipy complex by different concentrations of CA 15-3 in acetonitrile at pH 8.0 and λex = 340 nm. The quenching of luminescence intensity Tb-Bipy complex especially the electrical band at λem = 545 nm is typically used for determining the Carbohydrate Antigen (CA 15.3) in different body fluids. The dynamic range which is found for the assessment of Carbohydrate Antigen (CA 15.3) concentration is 0.1-15.4 U mL-1, and the limit of detection (LOD) and quantitation limit of detection (LOQ) are (0.109 and 0.57) U mL-1, respectively. This method was practical, simple and relatively free from interference effect. It was successfully applied to measure Carbohydrate Antigen (CA 15.3) in samples of human serum and from this method we can assess some biomarkers of cancer related diseases in human body.