The invention discloses a super-resolution biomolecular mass spectrometry imaging device and a working method thereof. The device is characterized by comprising: a laser, an optical modulator assembly, an optical element, a slide, a camera, a reflector and a mass spectrometer. The laser emitted by a laser is modulated by the optical modulator assembly, and is then focused on a target sample placed on the slide through the optical element, desorption ionization occurs to the target sample, then the sample enters the mass spectrometer to undergo detection, the optical modulator assembly consists of a coding aperture, by which the target sample at a corresponding pixel detection position is irradiated by laser, and the reflector and the camera are used for acquiring the targe sample information. The super-resolution biomolecular mass spectrometry imaging device provided by the invention adopts the mass spectrometry imaging technology based on the coding aperture and mass spectrometer, changes the inherent framework of existing lattice optical mechaical scanning, acquires super-resolution biomolecular microscopic mass spectrometry imaging, and has the advantages of high spatial resolution, high sensitivity, fast imaging speed and high signal-to-noise ratio.