Abstract: Objective To evaluate the effectiveness of bio-impedance spectroscopy (BIS) analysis for marginal tissue malignancy/benignancy identification during Breast-Conserving Surgery (BCS). Methods A multicenter parallel-group clinical trial was conducted with the patients who received breast conserving surgery in 2 hospitals. One hundred and fifty breast tissue specimens were collected and analyzed by BIS, frozen section biopsy and paraffin section biopsy respectively. Results were compared and statistical analysis was implemented. Results For BIS technique to identify malignant/benign marginal tissues, the sensitivity was 92.98%, and the specificity was 94.62%. The coherence of BIS results and frozen section biopsy results was significant, and the Kappa value was higher than 0.75. The mean measuring time of BIS was 2.63 min, which was shorter than frozen section biopsy (18.97 min) and paraffin section biospy (1.12 d). Conclusion BIS technique likely has high accuracy in tissue identification so has potential to be adopted as a real-time indicator for marginal tissue malignancy/benignancy identification during breast-conserving surgery.