Herein, tunable protein harmonic diffractive microlenses (PHDMs) have been constructed by femtosecond laser direct writing from bovine serum albumin. With excellent three-dimensional topography, PHDMs show distinct harmonic diffractive features, such as similarly good imaging performance with smaller thickness than refractive lenses and well controlled minus chromatic dispersion compared with simple diffractive lenses (∼5% focal length shift against ∼21% of the protein Fresnel zone plate with the same radius under light with wavelength 450-659 nm). Owing to the nature of protein molecules as "building blocks," the focal length of the PHDM can be facilely tuned within seconds by changing the pH value (focal length tunability of up to ∼20%).