Objective To observe the performance of hyperspectral non-mydriatic fundus camera prototype and its application on ocular fundus diseases. Methods The narrow band filters was inserted into the optical path of the Canon non-mydriatic retinal camera (CR-DGi). The image was converted to digital data by charge-coupled device (CCD), and then analyzed by hyperspectral data software. Twelve volunteers were examined by hyperspectral nonmydriatic fundus camera prototype to confirm the characteristic wavelength spectrums of ocular fundus diseases and the repeatability of prototype. Fifty-nine patients with ocular fundus diseases who underwent fluorescein angiography were also examined by hyperspectral non-mydriatic fundus camera prototype, to compared the images of prototype and fluorescein angiography. Results Each of the highest power of the light at the focus point and the power per unit were safe. 536, 547, 579 nm were selected as the specific retinal imaging spectrums and 608 nm as the specific choroidal imaging spectrum. The intra-observer and inter-observer reproducibility was equal or greater than 0.85. The correlation between hyperspectral non-mydriatic fundus camera prototype and fluorescein angiography in choroidal neovascularization patients were 0.782 and 0.833. Conclusions The hyperspectral nonmydriatic fundus camera prototype is safe and reliable. It shows pathological retinal and choroidal structures with specific spectrums. There are good prospects for the application in clinical diagnosis, especially for macular diseases.