Amplitude of low‐frequency fluctuation (ALFF) has been widely used for localization of abnormal activity at the single‐voxel level in resting‐state fMRI (RS‐fMRI) studies. However, previous ALFF studies were based on fast Fourier transform (FFT‐ALFF). Our recent study found that ALFF based on wavelet transform (Wavelet‐ALFF) showed better sensitivity and reproducibility than FFT‐ALFF. The current study aimed to test the reliability and validity of Wavelet‐ALFF, and apply Wavelet‐ALFF to investigate the modulation effect of repetitive transcranial magnetic stimulation (rTMS). The reliability and validity were assessed on multicenter RS‐fMRI datasets under eyes closed (EC) and eyes open (EO) conditions (248 healthy participants in total). We then detected the sensitivity of Wavelet‐ALFF using a rTMS modulation dataset (24 healthy participants). For each dataset, Wavelet‐ALFF based on five mother wavelets (i.e., db2, bior4.4, morl, meyr and sym3) and FFT‐ALFF were calculated in the conventional band and five frequency sub‐bands. The results showed that the reliability of both inter‐scanner and intra‐scanner was higher with Wavelet‐ALFF than with FFT‐ALFF across multiple frequency bands, especially db2‐ALFF in the higher frequency band slow‐2 (0.1992–0.25 Hz). In terms of validity, the multicenter ECEO datasets showed that the effect sizes of Wavelet‐ALFF with all mother wavelets (especially for db2‐ALFF) were larger than those of FFT‐ALFF across multiple frequency bands. Furthermore, Wavelet‐ALFF detected a larger modulation effect than FFT‐ALFF. Collectively, Wavelet db2‐ALFF showed the best reliability and validity, suggesting that db2‐ALFF may offer a powerful metric for inspecting regional spontaneous brain activities in future studies. Using multicenter RS‐fMRI datasets under EC and EO conditions and a single‐session rTMS modulation dataset, this study provides evidence of the superiority of Wavelet‐ALFF (especially db2‐ALFF) over FFT‐ALFF in improving reliability and validity and increasing statistical significance. Therefore, db2‐ALFF may offer a powerful metric for inspecting regional spontaneous brain activities in future studies.