A new method for rapidly acquiring fluorescence spectra from optical probes using an acousto‐optic tunable filter (AOTF) has been developed. The AOTF consists of a piezoelectric transducer bonded to a birefringent crystal. Acoustic waves are generated in the crystal by an applied radio frequency. For a given radio frequency, only a narrow band of optical frequencies will be diffracted. Unlike normal Bragg diffraction which can only be used with collimated light, the AOTF has a large angular aperture (up to 28 deg) and can therefore be coupled to an uncollimated broadband white light source, photomultiplier tube, or camera. Using the device and a xenon–mercury arc lamp, excitation and emission spectra of the fluorescentpH probe BCECF were acquired in 17 ms (spectral resolution=4 nm). Rapid wavelength switching or modulation of selected wavelengths was achieved at ∼105Hz. The device is also capable of spectral imaging with a spatial resolution of greater than 100 lines/mm.