Objective: In this study, we aimed to establish a frequency-specific ABR (fs-ABR) system via loudspeakers to assess the hearing improvement in ossiculoplasty intra-operatively and observe its efficiency and accuracy in predicting the long-term outcome. Setting Blackman-gated 1kHz tone-pips with 1ms, 2ms and 3ms duration were used in normal hearing (NH) subjects to calibrate the system and the standard ABR threshold and wave V latency for this system were established. All subjects would take four hearing tests: Pure tone audiometry (PTA) before and six-month after the surgery, fs-ABR under anesthesia before surgery and right after the ossicular chain reconstruction intra-operatively. PTA was used as the standard test to measure hearing. Bland-Altman analysis and linear correlation analysis were used to compare the agreement between PTA and fs-ABR results. Participants Forty-two conductive hearing loss (CHL) subjects. Results: For NH and CHL subjects in operating room before surgery, the fs-ABR threshold showed a high linear relation with the PTA results (r=0.88, P<.0001). For CHL follow up results: for 1ms group, PTAI showed a better correlation with fs-ABRI (r=0.67, P<.01) with the equation: PTAI=2.15*fs-ABRI-3.49; for 3ms group, PTA showed a better correlation with fs-ABR (r=0.76, P<.01) with the equation PTA=0.93*fs-ABR+3.48. Bland-Altman analysis showed no difference between PTA and fs-ABR in all above analysis. Eustachian tube malfunction would negatively affect the prediction efficacy, for subjects with normal ETF, the correlation between fs-ABRI and PTAI was even higher: PTAI=1.6*fs-ABRI+12.48 with r=0.77 (P=.0407<0.05). Conclusions This system could monitor the function of the reconstructed ossicular chain intra-operatively and predict the post-surgical 6-month hearing improvement efficiently and accurately. The average testing time for the fs-ABR was short, about 10 to 15 minutes. This system would serve as a promising tool clinically to help surgeons optimize the efficacy of ossiculoplasty. Besides, ETF should be taken into consideration as a risk factor that would negatively influence the hearing impairment.