Non-invasive methods for breast cancer detection in early stages may help to increase the survival rate of patients. This study aimed to evaluate the application of Anti-MUC1 antibody-based iron oxide nanoparticle (SPIONs-C595) which was assessed in vivo as a molecular imaging probe for breast cancer (MCF-7) detection using MRI. Nine groups of female NRC NU/Nu mice (each group of 3), 6 to 8 weeks old were used and MCF-7 cells were injected subcutaneously into both flanks of nude mice. After two weeks the mice received an intravenous injection of different concentrations of SPIONs-C595. The uptake ability of SPIONs-C595 on three-dimension (3D) macrostructure is exploited a modified hanging drop method using Prussian blue for MCF-7 cells. The iron content was measured in liver, kidney, spleen, and tumor. The MR imaging features and biodistribution of nanoprobe was also investigated. The MR images obtained from digested tumor after nanoprobe administration in different time-period revealed that enhancement of T1 and T2 relaxation time. Moreover, the storage stability test was shown great application and no sedimentation of nanoparticles within two months storage at 4°C. Additionally, great validation of SPIONs-C595 on the 3D spheroid of MCF-7 was observed. The biodistribution analysis showed that iron content of the spleen was more than the other studied organs. These results highlighted the feasibility of an in-vivo model for detection of breast cancer MUC1 expression. Current researches are ongoing to further enhancement of relaxation times for classification of MUC1 status using clinical specimens.