Powerline interference always disturbs recordings of biomedical signals. Numerous methods have been developed to reduce powerline interference. However, most of these techniques not only reduce the interference but also attenuate the 60 Hz power of the biomedical signals themselves. In the present study, we applied the S-transform, which provides an absolute phase of each frequency in a multi-resolution time-frequency analysis, to reduce 60 Hz interference. According to results from an electrocardiogram (ECG) to which a simulated 60 Hz noise was added, the S-transform de-noising process restored a power spectrum identical to that of the original ECG coincident with a significant reduction in the 60 Hz interference. Moreover, the S-transform de-noised the signal in an intensity-independent manner when reducing the 60 Hz interference. In both a real ECG signal from the MIT database and natural brain activity contaminated with 60 Hz interference, the S-transform also displayed superior merit to a notch filter in the aspect of reducing noise and preserving the signal. Based on these data, a novel application of the S-transform for removing powerline interference is established.