Multiphoton microscopy (MPM) has become an essential research tool in biomedicine. Current MPM systems predominantly rely on Ti:sapphire lasers provided tunable femtosecond pulses at 720–950 nm. To access the second biological transparency window (1000–1350 nm), complex optical parametric oscillators are typically required. urthermore, sources operating in the third biological transparency window (1600–1750 nm) are attracting significant attention for enhanced imaging depth. However, no ultrafast laser source simultaneously covering all three transparency windows exists, thus hindering the widespread application of MPM in life sciences. Here, we demonstrate a fiber-laser-based ultrafast source that generates four-color tunable pulses across 800–1650 nm, covering the full spectral range for multiphoton excitation. This source utilizes our proposed spectral selection technique via self-phase modulation (SESS). SESS ensures SPM-dominated spectral broadening, producing isolated spectral lobes. Filtering the outermost lobes will generate near-transform-limited pulses with broad wavelength tunability. Using this supercontinuum excitation source, we successfully realize label-free imaging of diverse biomedical specimens, validating the performance of MPM empowered by this novel driving source.