This study designed an audio signal acquisition system to achieve real-time detection of R peak for heart rate variability (HRV) analysis, substituting ECG-specific data acquisition board with sound card. It is proved that the R peak with low frequency can be gathered by sound card. The comparison with PowerLab signal acquisition system has shown that the acquisition system of R peak could accurately detect the R peak. The R peak detected by sound card and PowerLab signal acquisition system completely overlap each other, while RR intervals has no difference. The HRV analysis was accurate and reliable with the RR interval from sound card. This low cost, high performance, strong compatibility and easy upgraded acquisition system of R peak compared with the traditional data acquisition board has the value of practical uses and might have broad applications in the future.