An innovative biosensing design strategy was utilized for the first time to develop a label-free and practical immunosensor for ultrasensitive analysis of calreticulin (CRT), a potential biomarker of breast carcinoma. Unlike strategies used in literature, a conjugated polymer carrying carboxyl groups on its backbone was synthesized and utilized as an immobilization matrix. This immunosensor design strategy was composed of the attachment of anti-CRT antibodies onto iron oxide nanoparticles@gold nanoparticles@poly(3-thiophenemalonic acid) polymer (Fe3O4@AuNPs@PT(COOH)(2)) functionalized indium tin oxide (ITO) electrode through carbodiimide coupling reaction. EIS measurement results illustrated significant changes in charge transfer resistance (R-ct) on immunoreaction between anti-CRT and CRT proteins. The proposed immunosensor enabled the detection of CRT over the range of 0.02 pg/mL and 100 pg/mL with a correlation coefficient of 0.9994 under optimal conditions. The detection limit (LOD), the quantification limit (LOQ) and the sensitivity were also determined to be 8.2 fg/mL, 27.4 fg/mL and 270 k Omega fg mL(-1)cm(2), respectively. Furthermore, it also exhibited good repeatability, excellent reproducibility, long storage stability and reusability. Experimental results analyzed with T-test to compare the means of the repeatability and reproducibility data, and F-test to the distribution of the repeatability and reproducibility data. According to T and F-tests, the experimental results achieved from the studies with the suggested immunosensor were relatively satisfactory. Furthermore, this immunosensor was tested to measure CRT levels in human serum and spiked human serum samples, and acceptable recovery rates ranging from 94.05% to 106.62% were obtained. In sum, considering its fast and easy-to-fabricate properties, this new strategy offers a potential tool for CRT biomarker detection.