Autoantibodies targeting the β1-adrenergic receptor (AAB-β1) display agonist-like effects, which may have a pathogenic role in the progression of heart failure. Here, we used the electrophysiological recordings to explore the effects of AAB-β1-positive serum from Chinese patients with heart failure on the activity of the peak transient outward potassium current (Ito) and the end 50 ms steady-state potassium current (Iss) in mouse cardiac myocytes. We found that the AAB-β1-positive serum had no effect on the activity of Ito, but it produced a decrease in the currents of Iss. A low concentration of positive serum (1/100) had a small inhibitory effect on Iss. However, positive serum at 1 : 10, 1 : 20, and 1 : 50 significantly decreased Iss. The concentration-dependence analysis showed that the EC50 of AAB-β1-positive serum was 1/60.24 and its nH was 2.86. It indicated that the AAB-β1 could inhibit Iss in mouse cardiomyocyte in a concentration-dependent manner.