The invention relates to a protein matrix microlens array diffraction device and a preparation method thereof, and belongs to the technical field of biological detection equipment. The matrix of the protein matrix microlens array diffraction device is a protein crystal, and a microlens-shaped convex array is processed on a surface wherein a maximum side length L on the protein crystal is located.According to the d1 protein matrix microlens array diffraction device provided by the invention, the measurement is quantified into physical quantity that is easier to measure, such as the shape, distribution and spacing of a diffraction pattern, so that the detection accuracy and flexibility are improved. In the preparation method of the protein matrix microlens array diffraction device providedby the invention, the protein monocrystal is used as the matrix of the protein matrix microlens array diffraction device, thereby being more stable in a solution environment, longer in service life and wider in application range. In a microlens array processing method provided by the invention, a micro-protrusion is directly formed by using femtosecond laser processing, so that the processing is simple and convenient, and the thermal damage is small.