Abstract Background Hydroxychloroquine plays a role in antimalaria, immune modulation, and possible novel coronavirus-2019 activity in vitro. The unwanted effect on QT prolongation could lead to lethal arrhythmia. Recently, American College of Cardiology (ACC)　had announced to use a risk score system while treating patients with hydroxychloroquine. In this study, we investigated the possible risk factors of corrected QT (QTc) prolongation and validated the applicability of ACC risk score system in our cohort. Methods We retrospectively enrolled 4568 patients who had undergone long-term hydroxychloroquine. 167 patients had electrocardiography before and during hydroxychloroquine use. All baseline characteristics, laboratory data, comorbidities, and concurrent medications were all recorded. Results The majority (80.8%) of our cohort were female and the average age was 51.4 years old. The most common indication of hydroxychloroquine is an autoimmune disease (95.2%), and the average dosage was 315mg daily. In multivariable logistic regression, diabetes mellitus (OR, 9.286, 95% CI=2.026-45.22) and additional QTc prolonging medications (OR, 2.89, 95% CI=1.40-5.94) were stronger independent risk factors than ACC risk score (OR, 1.20, 95% CI=1.02-1.41) for QTc prolongation≧60 ms. In linear regression, comorbidities and QTc prolonging medications (Adjusted R2: 0.385) provided more accurate prediction of QTc response than the ACC risk score alone (Adjusted R2: 0.259). Conclusions For those patients with long-term hydroxychloroquine use, patients with DM and additional QTc prolonging medications were more susceptible to significant QTc prolongation. Patient’s baseline QTc interval, concurrent medications and comorbidities, rather than the ACC risk score, could be used to predict the response of QTc.