In order to solve such problems as difficulty in directly reading the spectral imaging of Rowland grating and the complex imaging algorithm for flat-field concave gratings, we design a spectrophotometer based on principle of the holographic concave grating for biochemical detection of specific protein analyzers. This structure is based on the Rowland grating, and uses two lenses as the transition elements to transform the curved surface imaging of Rowland grating to the flat-field spectrum. Meanwhile, we simulate and optimize the structure with the optical design software Zemax. The optimization results show that the spectrophotometer whose working band is 320-800 nm with the highest resolution of 0.5 nm and the overall resolution of 1.7 nm, can be directly read by the photodiode array and can achieve plane spectrum. The structural characteristics meet the measurement requirements of specific protein analyzers. The experimental results of the prototype are consistent with the simulation results, and the repeatability, accuracy, stability, and linearity of the instrument are all up to the industry standards.