Objective To investigate the specificity and targeting abilities of FITC-CSPLNTRFC peptide FITC-BCSP1 optical molecular probe on breast cancer cells Bcap-37. Methods Probe FITC-BCSP1 and negative control probe FITC-svBCSP1 were prepared by solid phase synthesis. MTT assay was used to determine the toxicity of the two probes on breast cancer cells Bcap-37. The specificity of the binding of FITC-BCSP1 probe to Bcap-37 cells was identified by flow cytometry and fluorescent inversed microscopy. The specificity and targeting abilities of FITC-BCSP1 probe for transplantation tumor in Bcap-37 cells tumor-bearing nude mice model were tested by optical molecular imager. Results The purity of the synthesized probe was more than 98%, identified by mass spectrometry and high performance liquid chromatography. FITC-BCSP1 and FITC-svBCSP1 probes had no effect on proliferation and activity of Bcap-37 cells at the concentrations of 50-300 mol/L (IR%≤30%). FCM results showed that the percentage of FITC-BCSP1-labeled cells in Bcap-37 cells was significantly higher than that in other cells (all P < 0.001), and the percentage of FITC-BCSP1-labeled Bcap-37 cells was significantly higher than that of the control group (P < 0.001). It was observed under inverted fluorescence microscope that there were a large number of fluorescent cells in FITC-BCSP1-labeled Bcap-37 cells, with a positive rate of 100%, while the positive rate of FITC-svBCSP1 group was only 1%. In vivo assay with Bcap-37 cells tumor-bearing nude mice model showed that FITC-BCSP1 probe could be specifically enriched in the transplantation tumor tissue. Conclusion The optical molecular probe FITC-BCSP1 has good specificity and targeting abilities on breast cancer cells and can be used in the early diagnosis of breast cancer.